Bioactive acetogenins and derivatives

ABSTRACT

Novel acetogenins isolated from Asimina triloba and Goniothalamus giganteus of the family Annonaceae and derivatives of those and other acetogenins are described. Bioactive cyclic formaldehyde acetal derivatives are prepared from Annonaceous acetogenins having 1,2-, 1,4- or 1,5-diols. A non-adjacent bis-tetrahydrofuran(THF) acetogenin is prepared from an unsaturated mono-THF acetogenin earlier isolated from Goniothalamus giganteus. The substantially pure acetogenins and acetogenin derivatives of the invention exhibit cytotoxicity to human solid tumor cell lines equipotent to adriamycin or orders of magnitude more potent than adriamycin, and they exhibit effective pesticidal activities.

FIELD OF INVENTION

This invention relates to the isolation, identification, derivatization,and use of natural products. More particularly this invention isdirected to substantially pure forms of cytotoxic Annonaceousacetogenins and certain ester and cyclized ether acetogenin derivatives.

BACKGROUND AND SUMMARY OF THE INVENTION

In recent years, an increased interest in the phyto-chemistry of theAnnonaceae has been sparked by the bioactivity-directed isolation of theantileukemic Annonaceous acetogenin, uvaricin, from Uvaria acuminata.Acetogenins are C₃₅ -C₃₉ compounds and typically contain two longhydrocarbon chains, one of which connects a terminal2,4-disubstituted-γ-lactone to a variable number of tetrahydrofuran(THF) rings. The hydrocarbon chains contain a number of oxygenatedmoieties which can be hydroxyls, acetoxyls and/or ketones. Recently,single-ring acetogenins containing double bonds, epoxide compounds whichlack THF rings and a compound lacking both epoxides and THF rings havebeen reported. These interesting newer compounds support the proposedpolyketide origin of the Annonaceous acetogenins and provide additionalclues to their biogenesis.

All acetogenins found to date contain multiple stereocentres, theelucidation of which often presents daunting stereochemical problems.Because of their waxy nature, the acetogenins do not produce crystalssuitable for X-ray crystallographic analysis. Relative stereochemistriesof ring junctions have typically been determined by comparison ofnatural compounds with synthetic model compounds and such methods haveproven to be invaluable with the acetogenins. Recently, the absolutestereochemistries of the carbinol centers of acetogenins have beendetermined with the help of synthetic model compounds and high fieldnuclear magnetic resonance (NMR) analysis of theirmethoxyfluoromethylphenylacetic acid (MPTA) esters (Mosher esters).

Most Annonaceous acetogenins are potently bioactive, but the mode ofaction of these compounds was unknown until Londerhausen et al.concluded in Pesticide Science, 33, 427-438 (1991), that they act toinhibit complex I of mitochondrial oxidative phosphorylation with anactivity several times that of rotenone.

In accordance with the present invention there are provided novel,cytotoxic acetogenins and acetogenin derivatives. One group ofacetogenins of this invention isolated from Asimina triloba arerepresented by the general formula

    CH.sub.3 (CH.sub.2).sub.3 --R.sub.2 --R.sub.1

wherein R₁, is a group of the formula ##STR1## and R₂ is a divalentgroup selected from the group consisting of ##STR2## and acetylatedderivatives thereof.

Another embodiment of this invention provides substantially purecompounds of the formula

    CH.sub.3 (CH.sub.2).sub.11 --R.sub.3 --(CH.sub.2).sub.5 R.sub.1 '

wherein R₁ ' is a group of the formula ##STR3## and R₃ is selected from##STR4## and acetylated derivatives thereof. The compound wherein --R₃-- is the divalent group (VI) is denominated goniocin, a naturallyoccurring acetogenin isolated from Goniothalamus giganteus. The compoundwherein --R₃ -- is the divalent group (VII) is prepared by epoxidationand subsequent acid-catalyzed cyclization of a previously reportedacetogenin, gigantetronenin (also isolated from Goniothalamusgiganteus), having the above formula wherein --R₃ -- is a divalent groupof the formula ##STR5##

Another aspect of this invention is directed to novel cyclicformaldehyde acetal derivatives of acetogenins having at least one1,2-diol, 1,4-diol and/or 1,5-diol moiety in their structure. Suchacetogenins include the art-recognized acetogenins bullatalicin,bullatanocin, squamocin, squamostatin A, gigantetrocin andgoniothalamicin. The cyclic intramolecular formaldehyde acetalderivatives are prepared by reacting the acetogenin starting materialwith a 2-3 fold molar excess of chlorotrimethylsilane and dimethylsulfoxide. Bis-cyclic formaldehyde acetals can be formed fromacetogenins having two independent 1,2-, 1,4-, or 1,5-diol moieties.Preparation of the cyclic acetals significantly facilitatesstereochemical structure elucidation. Moreover, the acetal derivativesshow enhanced cytotoxicities against certain human tumor cell lines. Theenhancement of the bioactive potencies and selectivities can permitimproved utilization of many natural acetogenins.

The present invention further provides pharmaceutical formulationscomprising an effective amount of an acetogenin compound for treating apatient having a tumor. As used herein, an effective amount of theacetogenin compound is defined as the amount of the compound which, uponadministration to a patient, inhibits growth of tumor cells, killsmalignant cells, reduces the volume or size of the tumors or eliminatesthe tumor entirely in the treated patient. Thus, the substantially purecompounds in accordance with this invention can be formulated intodosage forms using pharmaceutically acceptable carriers for oral orparenteral administration to patients in need of oncolytic therapy.

The effective amount to be administered to a patient is typically basedon body surface area, patient weight, and patient condition. Theinterrelationship of dosages for animals and humans (based on milligramsper meter squared of body surface) is described by Freireich, E. J., etal., Cancer Chemother. Rep., 50 (4): 219 (1966). Body surface area maybe approximately determined from patient height and weight (see e.g.,Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., pages 537-538(1970)). Preferred dose levels will also depend on the attendingphysicians' assessment of both the nature of the patient's particularcancerous condition and the overall physical condition of the patient.Effective anti-tumor doses of the present acetogenin compounds rangefrom about 1 microgram per kilogram to about 200 micrograms per kilogramof patient body weight, more preferably between about 2 micrograms toabout 100 micrograms per kilogram of patient body weight.

Effective doses will also vary, as recognized by those skilled in theart, dependant on route of administration, excipient usage and thepossibility of co-usage with other therapeutic treatments includingother anti-tumor agents, and radiation therapy.

The present pharmaceutical formulation may be administered via theparenteral route, including subcutaneously, intraperitoneally,intramuscularly and intravenously. Examples of parenteral dosage formsinclude aqueous solutions of the active agent, in an isotonic saline, 5%glucose or other well-known pharmaceutically acceptable liquid carrier.In one preferred aspect of the present embodiment, the acetogenincompound is dissolved in a saline solution containing 5% of dimethylsulfoxide and 10% Cremphor EL (Sigma Chemical Company). Additionalsolubilizing agents such as cyclodextrins, which form specific, moresoluble complexes with the present acetogenin compounds, or othersolubilizing agents well-known to those familiar with the art, can beutilized as pharmaceutical excipients for delivery of the acetogenincompounds. Alternatively, the present compounds can be chemicallymodified to enhance water solubility.

The present compounds can also be formulated into dosage forms for otherroutes of administration utilizing well-known methods. Thepharmaceutical compositions can be formulated, for example, in dosageforms for oral administration in a capsule, a gel seal or a tablet.Capsules may comprise any well-known pharmaceutically acceptablematerial such as gelatin or cellulose derivatives. Tablets may beformulated in accordance with conventional procedure by compressingmixtures of the active acetogenins and solid carriers, and lubricantswell-known to those familiar with the art. Examples of solid carriersinclude starch, sugar, bentonite. The compounds of the present inventioncan also be administered in a form of a hard shell tablet or capsulecontaining, for example, lactose or mannitol as a binder and aconventional fillers and tableting agents.

DETAILED DESCRIPTION OF THE INVENTION

Bullatacin is one of the most potent antitumor and pesticidalAnnonaceous acetogenins and was first reported and isolated from Annonabullata in 1989. The correct absolute configurations of the stereogeniccarbinol centers of bullatacin were recently established by ¹ H- and ¹⁹F-NMR spectral analysis of bullatacin's (S)- and (R)-Mosher ester[methoxy (trifluoromethyl)phenylacetate or MTPA] derivatives. Bullatacinhas shown potent in vivo antitumor activities, e.g., 67% tumor growthinhibition (TGI) at 50 μg/kg in athymic mice bearing A2780 human ovariancancer xenografts. Bullatacin likely inhibits cancer cell growth throughinhibition of mitochondrial electron transport systems to reduce the ATPlevels. Eleven Annonaceous acetogenins have been previously reportedfrom the ETOH extract of the stem bark of Asimina triloba. Directed bythe brine shrimp lethality test (BST), two related igsomericacetogenins, the bullatacin threo-trans-threo-trans-erythro from C-15 toC-24 isomers, bulletin (1), and bullanin (2), which have the thirdhydroxyl group at C-10 and C-30, instead of at C-4 have been discovered.

    __________________________________________________________________________     ##STR6##                                                                     compounds R.sub.1                                                                              R.sub.2 R.sub.3 R.sub.4 R.sub.5                                                                              R.sub.6                                                                              R.sub.7                __________________________________________________________________________    bullatin (1)                                                                            H      OH      OH      OH      H      H      H                      bullanin (2)                                                                            H      H       OH      OH      H      H      OH                     __________________________________________________________________________

Bullatin (1), a colorless wax, was obtained after HPLC separation fromthe more polar fractions of the 90% methanolic partition extract. Themolecular weight was determined as 622 by FAB mass spectrometry based onthe molecular ion peak (MH⁺) m/z 623. The molecular formula was thendeduced as C₃₇ H₆₆ O₇ by the high resolution FAB mass spectrum whichgave the molecular ion at m/z 623.4865 corresponding to the calculatedexact mass of 623.4887. The IR spectrum of 1 showed characteristicabsorptions of the α,β-unsaturated γ-lactone (1748 cm⁻¹, C═O) andhydroxyl (3442 cm⁻¹, OH) functional groups. The presence of theα,β-unsaturated γ-lactone moiety was confirmed by the ¹ H NMR resonancepeaks (Table 1) at δ6.99 (1, H-35), 5.00 (11, H-36) and ¹³ C NMRresonances (Table 1) at δ173.83 (C-1), 134.21 (C-2), 148.85 (C-35),77.43 (C-36), and 19.27 (C-37). Spectral comparisons of 1 withbullatacin, the 4-hydroxylated isomer, indicated that the H-3 signalsappeared as a multiplet peak at δ2.26 (tt 2H) in the ¹ H NMR spectrum of1 instead of an AB spin system corresponding to the H-3a and H-3b protonresonance in the ¹ H NMR spectrum of bullatacin, and the upfield shiftof proton signals for H-35 and H-36 of compound 1 suggested that the4-OH group was absent in the structure of bullatin (1).

Careful examination of the ¹ H NMR spectrum of 1 showed the typicalproton resonances of the adjacent bis THF acetogenins. The threemultiplets at δ3.41, 3.87, and 3.93 integrated for one proton, fourprotons and one proton, respectively, on oxygen-bearing carbons, and the¹³ C NMR resonances of these methine carbons at δ71.31, 73.99, 82.52,82.33, 82.81 and 83.14 indicate the presence of the structural unit ofan adjacent bis-THF moiety with two flanking OH groups. ¹ H and ¹³ C -spectral comparisons of 1 with bullatacin suggested that the relativestereochemical relationship of 1 across the bis-THF skeleton from C-15through C-24 is threo-trans-threo-trans-erythro, and, thus, 1 belongs tothe bullatacin type acetogenins. This conclusion was confirmed by theexpected downfield shift of these methine proton resonances and theirlocations at δ

                  TABLE 1                                                         ______________________________________                                        .sup.1 H (500 MHz) and .sup.13 C (125 MHz) NMR Data of 1 and 1a                                                 .sup.13 C-NMR                               H/C No. .sup.1 H-NMR of 1                                                                          .sup.1 H-NMR of 1a                                                                         of 1                                        ______________________________________                                         1                   --           173.83                                       2                   --           134.21                                       3      2.26 tt (7.6, 1.5)                                                                         2.26 tt (7.6, 1.5)                                                                          22.73-31.94                                 4      1.55 m       1.40-1.80 m   22.73-31.94                                 9, 11  1.43 m       1.40-1.80 m   37.423 37.45                               10      3.59 m       4.85 m        71.85*                                     14, 25  1.40 m       1.40-1.80 m   33.31, 33.46                               15      3.41 m       4.85 m        73.99                                      16      3.87 m       3.98 m        83.14**                                    17, 18, 21                                                                            1.98 m, 1.64 m                                                                             1.98 m, 1.64 m                                                                              22.73-31.94                                19, 20  3.87 m       3.91 m        82.52, 82.33                               22      1.81 m, 1.89 m                                                                             1.80 m, 1.89 m                                                                              22.73-31.94                                23      3.93 m       3.98 m        82.81**                                    24      3.87 m       4.92 m        71.31*                                      5-8, 12-13,                                                                          1.20-1.50 m  1.20-1.80 m   22.73-31.94                                26-33                                                                         34      0.878 t (7.0)                                                                              0.878 t (7.0)                                                                               14.18                                      35      6.99 q (1.5) 6.99 q (1.5) 148.85                                      36      5.00 qq (6.8, 1.8)                                                                         5.00 qq (6.8, 1.7)                                                                          77.43                                      37      1.41 d (7.0) 1.41 d (7.0)  19.27                                      15-OAc               2.08 s                                                   24-OAc               2.05 s                                                   10-OAc               2.04 s                                                   ______________________________________                                         *, **indicate assignments which may be interchangable.                   

                                      TABLE 2                                     __________________________________________________________________________     ##STR7##                                                                     R        A      B      C      D       E      F                                __________________________________________________________________________    H        (427)  225    311    (381)          171                              (EIMS)   409 (a)       293 (a)                                                                              363 (a)                                                  391 (a)       275 (a)                                                                              345 (a)                                                  373 (a)              327 (a)                                         TMS      643    297    455    (525)   595    243                              (EIMS)   553 (b)       365 (b)                                                                              435 (b) 505 (b)                                                        275 (b)        415 (b)                                                                       397 (a)                                 D.sub.9 -TMS                                                                           (670)  306    473    (543)   613    252                              (EIMS)   571 (c)       374 (c)                                                                              444 (c) 514 (c)                                          472 (c)       275 (c)                                                                              345 (c) 415 (c)                                          373 (c)              327 (c) 397 (a)                                 __________________________________________________________________________     (a): loss of H.sub.2 O (m/z 18);                                              (b): loss of TMSiOH (m/z 90);                                                 (c): loss of d.sub.9 -TMSiOH (m/z 99).                                   

3.91, 3.98, 4.85, and 4.92 in the ¹ H NMR spectrum of bullatintriacetate (1a).

The existence of three OH groups was determined by successive losses ofthree water molecules (m/z 18×3) in the EIMS spectrum of 1 (Table 2) andthe three single acetgate methyl proton signals, at δ2.04, 2.05 and2.08, in the ¹ H NMR spectrum of bullatin triacetate (1a) (Table 1). Inaddition to the two flanking OH groups on each side of the THF ringsystem there had to be a third hydroxyl in this structure and it couldnot be located at C-4. In the ¹ H NMR spectrum of 1, the methine protonon the carbon to which the third hydroxyl group was attached appeared atδ3.59 and showed its only cross peak with the complex of methyleneproton signals at δ1.43 in the ¹ H- ¹ H 2D homonuclear correlation(COSY) spectrum, indicating that this third OH group was locatedsomewhere in the aliphatic chain.

The placement of the adjacent bis-THF ring at C-16 through C-23 and thethree OH groups at C-10, C-15 and C-24 in the aliphatic chain wasestablished by the analysis of the EIMS spectra of the tri-trimethylsilyl (tri-TMS) and tri-deutero-trimethylsilyl (d₉ -tri-TMS) derivativesof 1 (Table 2). The crucial fragment peaks at m/z 297 in the EIMSspectra of the tri-TMS derivative and m/z 306 in the EIMS spectra of thed₉ -tri-TMS derivative of 1, due to the cleavage of the bond betweenC-10 and C-11, indicated that the third OH group was located at the C-10position. This conclusion was further confirmed by the exact massdetermination of these fragment ions of the tri-TMS and d₉ -tri-TMSderivatives, i.e., for the m/z 297 ion peak the exact mass 297.1886 wasfound corresponding to the formula, C₁₆ H₂₉ SiO₃ (calcd. 297.1886), andfor the m/z 306 ion the exact mass of 306.2451 was found correspondingto the formula of C₁₆ H₂₀ D₉ SiO₃ (calcd. 306.2451). The full assignmentof proton resonance signals was made by analysis the ¹ H--¹ H 2D NMR(COSY) spectrum of 1. Thus, the structure of bullatin was concluded tobe as indicated above.

Bullanin (2) was obtained in a waxy form. The IR, ₁ H and ¹³ C NMR(Table 2), and ¹ H--¹ H correlation (COSY) spectra of bullanin were verysimilar to bullatin (1). The existence of three OH groups was indicatedby the broad IR absorption peak at 3442 cm⁻¹ and the three acetatemethyl resonances at δ2.04, 2.05 and 2.08 in the ¹ H NMR spectrum ofbullanin triacetate (2a) (Table 3). The appropriate proton and carbonresonance signals were attributed to the α,β-unsaturated δ-lactonemoiety and the adjacent bis-THF ring system, as the ¹ H and ¹³ C NMRspectra of bullanin showed little differences from those of bullatin(1).

The low resolution FAB mass of bullanin gave the [MH⁺ ]peak at 623,indicating a molecular weight of 622. This was confirmed by themolecular ion peak at m/z 623.4915 in the high resolution FAB massspectrum corresponding to the molecular formula C₃₇ H₆₆ O₇ (calcd.623.4887). Thus, bullanin was identified as another isomer ofbullatacin, having the same carbon skeleton and relativestereochemistries as bullatin (1) but with a different location of thethird OH group. The relative stereochemistry at the six chiral centersfrom C-15 through C-24 across the bis-THF ring of bullanin was confirmedas threo-trans-threo-trans-erythro by the typical downfield shifts andplacements of these oxygen-bearing methine proton signals in the bis-THFrings in the ¹ H NMR spectrum of bullanin triacetate (2a) (Table 3).

The EIMS diagnostic fragment peaks of the tri-TMS and d₉ -tri-TMSderivatives of bullanin (Table 4) determined the positions of theadjacent bis-THF ring moiety and the third OH group. Careful analysis ofthe EIMS fragmentation ions of the tri-TMS and d₉ -tri-TMS derivativesled us to conclude that, like bullatin, the bis-THF ring with twoflanking OH

                  TABLE 3                                                         ______________________________________                                        .sup.1 H (500 MHz) and .sup.13 C (125 MHz) NMR Data of 2 and 2a                                                 .sup.13 C-NMR                               H/C No. .sup.1 H-NMR of 2                                                                          .sup.1 H-NMR of 2a                                                                         of 2                                        ______________________________________                                         1      --           --           173.83                                       2      --           --           134.23                                       3      2.26 tt (7.5, 1.5)                                                                         2.26 tt (7.5, 1.5)                                                                          22.80-29.76                                 4      1.54 m       1.54 m        22.80-29.76                                14, 25  1.39 m       1.46-1.64 m   33.33, 32.27                               15      3.40 m       4.86 m        74.11                                      16      3.85 m       3.98 m        83.25**                                    17, 18, 21                                                                            1.98 m, 1.63 m                                                                             1.95 m, 1.78 m                                                                              22.80-29.76                                19, 20  3.85 m       3.89 m        82.52, 82.26                               22      1.90 m, 1.81 m                                                                             1.95 m, 1.78 m                                                                              22.80-29.76                                23      3.93 m       3.98 m        82.75**                                    24      3.85 m       4.91 m        71.28*                                     29, 31  1.40 m       1.46-1.64 m   37.17, 37.34                               30      3.58 m       4.86 m        71.87*                                      5-13,  1.22-1.72 m  1.20-1.38 m   22.80-29.76                                26-27,                                                                        31-33                                                                         34      0.907 t (7.0)                                                                              0.907 t (7.0)                                                                               14.14                                      35      6.99 q (1.5) 6.99 q (1.5) 148.79                                      36      5.00 qq (7.0, 1.5)                                                                         5.00 qq (7.0, 1.5)                                                                          77.41                                      37      1.41 d (7.0) 1.41 d (7.0)  19.26                                      15-OAc               2.08 s                                                   24-OAc               2.05 s                                                   29-OAc               2.04 s                                                   ______________________________________                                         *, **Indicate assignment which may be interchangable.                    

                                      TABLE 4                                     __________________________________________________________________________     ##STR8##                                                                     R     A     B    C     D    E     F     G    H                                __________________________________________________________________________    H     295   (327)      (257)                                                                              (435) 187         565                             (EIMS)                                                                              277 (a)                                                                             (309)      239 (a)                                                                            417 (a)                                                                             169 (a)    (547)                                        291 (a)    221 (a)    151 (a)    (529)                                                                         511 (a)                          TMS   367   (471)                                                                              437   (401)                                                                              507   331   159  781                              (EIMS)      381 (b)                                                                            347 (b)                                                                             311 (b)                                                                            417 (b)                                                                             241 (b)    (691)                                        291 (b)                                                                            329 (a)                                                                             293 (a)                                                                            399 (a)                                                                             151 (b)    601 (b)                          d.sub.9 -TMS                                                                        376   (489)                                                                              (446) 419  516   349   168  (808)                            (EIMS)                                                                              277 (c)                                                                             390 (c)                                                                            347 (c)                                                                             320 (c)                                                                            417 (c)                                                                             250 (c)                                                 291 (c)                                                                            329 (a)    399 (a)                                                                             151 (c)                                     __________________________________________________________________________     (a): loss of H.sub.2 O (m/z 18);                                              (b): loss of TMSiOH (m/z 90);                                                 (c): loss of d9TMSiOH (m/z 99).                                          

groups was also located at C-15 through C-24. The hypothesis that thethird OH group was placed at C-30 was confirmed by the crucialdiagnostic mass ion at m/z 159 in the EIMS spectrum of the tri-TMSderivative (high resolution EIMS found: m/z 159.1203, calcd. 159.1205for C₈ H₁₉ SiO) due to cleavage between C-29 and C-30. Confirmingsupport for the C-30 hydroxyl was made by the mass ion at m/z 168 in theEIMS spectrum of the d₉ -tri-TMS derivative (high resolution EIMS found:m/z 168.1771, calcd. 168.1770 for C₈ H₁₀ D₉ SiO). The proton signalassignments (Table 3) were made by detailed analysis of the ¹ H--¹ Hcorrelated 2D spectrum (COSY). From these data, the structure ofbullanin (2) was concluded to be as that shown above.

Bioactivity data obtained with 1 and 2 are summarized in Table 5. Eachof these acetogenins were highly toxic to the brine shrimp larvae,showed highly potent cytotoxicities against three solid tumor celllines, A-549 (human lung carcinoma), MCF-7 (human breast carcinoma) andHT-29 (human colon adenocarcinoma), and were several orders of magnitudemore cytotoxic than the standard reference, adriamycin. The highlypotent cytotoxicities of bullatacin isomers 1 and 2 suggested that thepresence of a third OH group at either C-4, C-10, C-28, C-29 or C-30 isresponsible for the enhancement of activity. Bullatin (2) approaches thelevel of potencies of bullatacin (2). In mitochondrial inhibitionassays, using rat liver mitochondria, bullatacin, bullatin (1) andbullanin (2) all show high activity with bullanin (2) showing slightlyhigher potencies than bullatacin.

Experimental Details for Isolation and Characterization of Compounds 1and 2.

                                      TABLE 5                                     __________________________________________________________________________    Bioactivity Data of Acetogenins 1-7.                                                  BST     A-549   MCF-7   HT-29                                         Compounds.sup.e                                                                       LC.sub.50 (μg/ml)                                                                  LC.sub.50 (μg/ml)                                                                  LC.sub.50 (μg/ml)                                                                  LC.sub.50 (μg/ml)                          __________________________________________________________________________    1       4.0 × 10.sup.-3                                                                 9.39 × 10.sup.-6                                                                8.33 × 10.sup.-6                                                                3.78 × 10.sup.-6                        2       6.0 × 10.sup.-3                                                                 .sup. 3.11 × 10.sup.-14                                                          3.22 × 10.sup.-14                                                              .sup. 4.77 × 10.sup.-12                 3       4.6 × 10.sup.-3                                                                 7.99 × 10.sup.-9                                                                9.57 × 10.sup.-9                                                                <10.sup.-12                                   4       5.7 × 10.sup.-3                                                                 3.58 × 10.sup.-9                                                                <10.sup.-12                                                                           <10.sup.-12                                   5       4.9 × 10.sup.-3                                                                 3.29 × 10.sup.-7                                                                2.74 × 10.sup.-9                                                                <10.sup.-12                                   Adriamy-                                                                              2.57 × 10.sup.-1                                                                1.04 × 10.sup.-4                                                                1.76 × 10.sup.-2                                                                1.53 × 10.sup.-4                        cin                                                                           __________________________________________________________________________     Compounds 1-5 were bioassayed as the 2/4cis and trans mixtures; Adriamyci     was included as expected variations between runs are up to two orders of      magnitude as evidenced by adriamycin data on file.                            BST = brine shrimp lethality test                                             A-549 = cytotoxicity to human lung carcinoma                                  MCF-7 = cytotoxicity to human breast carcinoma                                HT-29 = cytotoxicity to human colon adenocarcinoma                       

Bioassays

The brine shrimp (Artemia salina Leach) test (BST) was performed asmodified to determine LC₅₀ values in μg/ml. Seven-day in vitrocytotoxicity tests against human tumor cell lines were carried out atthe Purdue Cancer Center, using standard protocols for A-549 (human lungcarcinoma), MCF-7 (human breast carcinoma) and HT-29 (human coloncarcinoma) with adriamycin as a positive control. The reported ED₅₀values in μg/ml (Table 5) were tabulated from the same run in order tofacilitate comparison for the SAR's.

Plant Material

The bark of Asimina triloba (L.) Dunal was collected from stands growingwild at the Purdue Horticultural Research Farm, West Lafayette, Ind.,U.S.A. The identification was confirmed by Dr. George R. Parker,Department of Forestry and Natural Resources, Purdue University. Avoucher specimen of the bark is preserved in the pharmacognosyherbarium.

Extraction and Purification of Acetogenins

The air-dried pulverized stem bark (15 kg) was extracted exhaustively(12 days) at room temperature with 95% EtOH (451×4) and vacuumevaporated to yield extract F001 (1645 g) which was partioned between H₂O (41) and CH₂ Cl₂ (41×5), giving a water soluble fraction (F002, 5 g),CH₂ C₂ soluble fraction (F003, 1560 g) and an insoluble interface (F004,80 g). F003 was further partitioned between hexane (41) and 90% MeOH aqsoln (41×5) and yielded the MeOH fraction (F005, 650 g) and the hexanefraction (F006, 905 g).

Directed by the BST bioassay, the most bioactive fraction, F005 (BSTLC₅₀ 7,151×10⁻¹ μg/ml) (200 g) was further fractionated by open columnchromatography on silica gel (8 Kg, 60-200 mesh), eluting withhexane-EtOAc and EtOAc-MeOH gradients; 12 pools were made from thecollected fractions according to their TLC patterns and evaluated by theBST bioassay. The most active pools (P₇ -P₉) were combined (20 g) andsubjected to further repeated separation by silica gel (1000 g, 230-400mesh) column chromatography eluted with MeOH-CHCl₃ gradients. Furtherpurifications of the most bioactive fractions F11-12 (1.2 g, BST LC₅₀3.5×10⁻² mg/ml) were carried out on Chromatotron plates (2 mm thick),eluted with hexane-CH₂ CHl₂ - MeOH (30:20:0-5). A white wax from thehexane-CH₂ Cl₂ -MeOH elutions was further resolved by HPLC eluted with10% THF in MeOH-hexane gradients (5-15%), MeOH (0-1%) in the mixture ofhexane and EtOAc (2:1) and acetone in CHCl₃ (0-10%) to yield acetogenins(1-2).

Bullatin (1)

Compound (1) was obtained as a colorless wax (12.5 mg). [α]D+7.5° (c=0.4mg/ml, EtOH). Uv λ_(max) EtOH nm:213 (log e=2.34). Ir V_(max) ^(film)cm⁻¹ :3442(OH), 2971, 2849, 1748 (C═O), 1458, 1186, 1051. FAB HR-MS(glycerol) m/z:623.4865 ([M+H])⁺, found), (required 623.4887, Calcd. forC₃₇ H₆₆ O₇). EIMS, EIMS (triTMSi derivative), and EIMS (tri-d₉ -TMSiderivative) m/z see Table 2. ¹ H NMR (500 MHz, CDCl₃); see Table 1. ¹³ CNMR (125.75 MHz, CDCl₃); see Table 1.

Bullatin Triacetate (1a)

Treatment of compound (1) (2 mg) with Ac₂ O-pyridine (at roomtemperature, overnight) and subsequent workup gave 1a as a colorlesswax. ¹ H NMR (500 MHz, CDCl₃); see Table 1.

Bullanin (2)

Compound (2) was obtained as a colorless wax (10 mg). [α]D+28° (c=0.5mg/ml, EtOH). Uv λ_(max) EtOH nm:220 (log e=2.42). Ir γ_(max) ^(film)cm^(-1:) 3442 (OH), 2927, 2852, 1747 (C═O), 1459, 1320, 1193, 1070. FABHRms (glycerol) m/z:623.4915 ([M+H]⁺, found), (623.4887, Calcd. for C₃₇H₆₆ O₇). EIMS, EIMS (triTMSi derivative), and EIMS (tri-d₉ -TMSiderivative) m/z see Table 4. ¹ H NMR (500 MHz, CDCl₃); see Table 3. ¹³ CNMR (125.75 MHz, CDCl₃); see Table 3.

Bullanin triacetate (2a)

Treatment of compound (2) (2 mg) with Ac₂ O-pyridine (at roomtemperature, overnight) and subsequent workup gave 2a as a colorlesswas. ¹ H NMR (500 MHz, CDCl₃); see Table 3.

Asimicin was the first acetogenin isolated from the seeds and stem barkof the North American paw paw tree, Asimina triloba Dunal (Annonaceae).Asimicin has been reported as exhibiting highly potent antitumor andpesticidal activities. Further studies of A. triloba stem bark has ledto the discovery of additional novel bioactive acetogenins, includingthe very active adjacent bis-tetrahydrofuran (THF) compound, trilobacin.The absolute stereochemistry of asimicin has now been defined, and themechanism of action of asimicin and other Annonaceous acetogenins is viainhibition of NADH: ubiquinone oxidoreductase (complex I) inmitochondrial electron transport systems.

Further activity-directed fractionation of the ethanolic extract of thestem bark, using the brine shrimp lethality test (BST) to monitorfractionation, has revealed three novel adjacent bis-THF acetogenins,asimin (3), asiminacin (4) and asiminecin (5). Compounds 3-5 arestructurally identified as asimicin isomers with the C-4 hydroxyl groupmoved to the C-10, C-28 and C-29 positions, respectively. These threecompounds are nearly identical in their ¹ H- and ¹³ C- NMR spectra. Thedeterminations of the hydroxyl group positions were based on the massspectral analysis of their tri-TMSi and tri-d₉ -TMSi derivatives. Cellculture data showed these compounds have highly potent in vitrocytotoxicities against three human tumor cell lines in cytotoxicitytests.

Isolation of asimin, asiminacin and asiminecin

In searching for new bioactive acetogenins from the F005 fraction, whichwas partitioned from the EtOH extract of the stem bark, the more polarcolumn fractions from the most active pools (P7-P9) were investigated.The fraction sample was subjected to open column and chromatotronchromatography with gradient elutions using MeOH and CH₂ Cl₂ and 0-5% ofMeOH in a mixture of CHCl₃ and hexane (2:3). Fractions 14-19 on TLCplates exhibited a unique spot which failed to be further resolved onadditional chromatotron plates. At first this material was considered asa single compound. The ¹ H-NMR spectrum appeared to represent a singlecompound, but expansion of the proton signals of the terminal methylgroups showed an overlap of several compounds. A satisfactory separationwas then achieved by

    __________________________________________________________________________     ##STR9##                                                                     Compounds       R.sub.1                                                                            R.sub.2                                                                             R.sub.3                                                                           R.sub.4                                                                            R.sub.5                                                                            R.sub.6                              __________________________________________________________________________    Asimicon        OH   H    OH   OH   H    H                                    Asimin (3)      H    OH   OH   OH   H    H                                    Asimin triacetate (3a)                                                                        H    OAc  OAc  OAc  H    H                                    Asiminacin (4)  H    H    OH   OH   OH   H                                    Aciminacin triacetate (4a)                                                                    H    H    OAc  OAc  OAc  H                                    Asiminecin (5)  H    H    OH   OH   H    OH                                   Asiminecin triacetate (5a)                                                                    H    H    OAc  OAc  H    OAc                                  __________________________________________________________________________

using HPLC. Four separated HPLC peaks (A-D) were obtained by using 8 μmnormal phase silica gel HPLC columns with gradient elution of 8-12% of amixture of MeOH and tetrahydrofuran (9:1) in hexane. Peak C was in pureform and gave compound 5. Further resolution of peak A wassatisfactorily achieved to give compounds 3 and 4 in pure form bydecreasing the MeOH percentage to 5-10% and increasing the ramping time.

Compound 3 was obtained as a colorless wax. A CIMS (isobutane) peak atm/z 623 indicated the molecular weight of 622. HR FABMS (glycerol) foundthe molecular ion peak at m/z 623.4907 which closely matched the exactmass 623.4887 calculated for the molecular formula C₃₇ H₆₆ O₇. An IRabsorption band at 1751.6 cm-1 and UV λ_(max) at 215 nm and the ¹ H-NMRspectrum peaks (Table 6) at 66.99 (q, H-35), 5.00 (qq, H-36), as well as¹³ C-NMR resonances (Table 6) at δ173.80 (C-1) 134.20 (C-2), 148.82(C-35), 77.41 (C-36), 19.27 (C-37), and 25.20 (C-3), suggested thepresence of an α,β-unsaturated λ-lactone ring as previously reported intrilobacin and asimicin. Spectral comparisons of compound 3 and asimicinclearly indicated that the H-35 and H-36 proton signals and the C-35 andC-36 carbon resonances of 3 were shifted slightly upfield and the C-1and C-2 signals were shifted downfield; the H-3a and N-3b peaks,characteristic in the ¹ H-NMR spectrum of asimicin were missing in 3,and the appearance of a multiplet peak at δ2.26 (tt, 2H) suggested theabsence of a hydroxyl group at the C-4 position. Therefore, the presenceof the typical subunit, fragment A (as illustrated on page 3 line 31),was substantiated.

The integrations for four protons in the multiplet signal at 3.85 ppm(H-16, 19, 20, and 23) and for two protons in the broad peak at 3.40 ppm(H-15 and 24), in the ¹ H-NMR spectrum (Table 6) of 3, indicated that 3is an adjacent bis-THF-acetogenin. The ¹³ C-NMR spectrum (Table 6),likewise, showed three pairs of closely located

                                      TABLE 6                                     __________________________________________________________________________    NMR Data for Asimin (3) and Its Triacetate derivative (3a)                             .sup.1 H-NMR      .sup.13 C-NMR                                               (500 MHz, CDCl.sub.3, δ in ppm, J                                                         (125.75 MHZ, CDCl.sub.3,                                    in Hz)            δ in ppm)                                    H/C No.  3        3a       >3                                                 __________________________________________________________________________     1       --       --       173.80                                              2       --       --       134.20                                              3       2.26 tt (7.8, 1.6)                                                                     2.26 tt (7.8, 1.6)                                                                      31.94-22.73                                        9, 11   1.44 m   1.46-1.64 m                                                                             37.43, 37.45                                      10       3.59 m   4.96 m    71.84                                             14, 23   1.65 m   1.46-1.64 m                                                                             33.39, 33.46                                      13, 24   3.40 m   4.86 m    73.97, 74.08                                      16, 23   3.85 m   3.99 m    83.05, 83.17                                      17a, 18a, 21a, 22a                                                                     1.98 m   1.95 m    31.94-22.73                                       17b, 18b, 21b, 22b                                                                     1.65 m   1.78 m    31.94-22.73                                       19, 20   3.85 m   3.91 m    81.76 81.81                                        4-8, 12-13, 26-33                                                                     1.24-1.60 m                                                                            1.20-1.38 m                                                                             31.94, 29.66, 29.64, 29.57,                                                   29.37, 29.30, 29.14, 29.03,                                                   29.02, 28.39, 27.41, 25.73,                                                   25.70, 25.64, 25.20 22.73                         34       0.878 t (7.0)                                                                          0.879 t (7.0)                                                                           14.18                                             35       6.99 q (1.5)                                                                           6.99 q (1.5)                                                                           148.82                                             36       5.00 qq (6.8, 1.7)                                                                     5.00 qq (6.8, 1.7)                                                                      77.41                                             37       1.41 d (6.5)                                                                           1.41 d (7.0)                                                                            19.27                                             15-OAc            2.08 s                                                      24-OAc            2.08 s                                                      10-OAc            2.04 s                                                      __________________________________________________________________________

resonance peaks, at δ83.05 and 83.17, 81.76 and 81.81, and 73.97 and74.08, which confirmed the presence of a moiety having adjacent bis-THFrings with two flanking hydroxyl groups. These proton and carbon signalswere very similar to those observed with asimicin. In order to determinethe relative stereochemistry between the adjacent chiral centers in thisfragment, a triacetate derivative (3a) was made. The ¹ H-NMR data of 3a(Table 6) showed three separated oxygen-bearing methine signals; two ofthese peaks were integrated for two protons each at the resonances ofδ3.91 and 3.99 arising from H-19, 20 and H-16, 23, respectively; thethird signal, which integrated for three oxygen-bearing methine protons,was located at δ4.86 and had shifted downfield due to the acetylation ofthree hydroxyl groups; two of the hydroxyls were those flanking bothsides of the bis-THF ring system. This chemical shift pattern closelymatched the relative stereochemistry of a threo-trans-threo-trans-threosystem which is identical to that of asimicin and is represented byfragment B (as included in structures III-V on page 3, lines 6-20). Aslight separation of the three pairs of oxygen-related carbons hintedthe influence of the third hydroxyl group located near the bis-THF ringsystem.

The presence of the three hydroxyl groups in 3 was confirmed by the IRabsorption at 1751.6 cm-1 and the proton signals at δ3.40 (2H) and 3.59(¹ H). In the CIMS spectrum (Table 7), the observation of a series offragments at m/z 605,587, and 569, due to successive losses of threemolecules of water, further confirmed the presence of three hydroxylgroups. Additional evidence was provided by the three acetate methylsignals at δ2.04 (3H) and 2.08 (6H) in the ¹ H-NMR spectrum of 3a andthe fragment peaks at m/z 689, 629, and 569 arising from the successivelosses of three acetates (m/z 60) from the molecular ion at m/z 749 inthe CIMS spectrum of 3a. Two hydroxyl groups were those flanking the THFring, and the third hydroxyl, which

                                      TABLE 7                                     __________________________________________________________________________     ##STR10##                                                                    R    M.sup.+ /MH.sup.+                                                                    A     B     C     D    E     F    G                               __________________________________________________________________________    H    623          225   311 (a)    241*       171                             (CIMS)                                                                             605 (a)            293 (a)    223* (a)                                        587 (a)            275 (a)                                                    569 (a)                                                                  TMS  838*   643   297   455        313*  595  243                             (EIMS)                                                                             748 (b)                                                                              553 (b)                                                                             207* (b)                                                                            365 (b)    223* (b)                                                                            505 (b)                                                                            153* (b)                             658* (b)                                                                             463* (b)    275* (b)         415 (b)                                   568* (b)                                                                             273* (b)                                                          D.sub.9 -TMS                                                                       865*   670   306   473   543  322*  613  252                             (EIMS)                                                                             766 (c)                                                                              571 (e)                                                                             207* (c)                                                                            374 (c)                                                                             444 (c)                                                                            223* (c)                                                                            514 (c)                                                                            153* (c)                             667 (c)                                                                              472 (c)     275 (c)                                                                             345 (c)    415 (c)                                   568 (c)                                                                              373* (c)                                                          __________________________________________________________________________     (a): loss of H.sub.2 O (m/z 18);                                              (b): loss of TMSi (m/z 90);                                                   (c): loss of d.sub.9 -TMSi (m/z 99);                                          *weak intensity.                                                              Diagnostic mass fragmentation ions of asimin (3) and its triTMSi and          trid.sub.9 -TMSi derivatives.                                            

exhibited its methine proton signal at δ3.59 and its carbon resonance atδ71.84, was predicted to be nearby in the aliphatic chain.

Having determined the presence of fragments A and B and the threehydroxyl groups; the remaining portions of the structure of compound 3consisted simply of two aliphatic chains. To establish the location ofthese fragments and hydroxyl groups along the aliphatic chains, thetriTMSi and tri-d₉ -TMSi derivatives were prepared. Analysis of the EIMSspectra (Table 7) of these derivatives determined that the adjacentbis-THF ring system was located at C-16 to C-23 and the three hydroxylgroups are at C-10, C-15 and C-24. Thus, the structure of compound 3 wasestablished as that illustrated above, and 3 was given the name ofasimin.

Compound 4 was isolated as a wax in a similar amount as 3. The CIMS(isobutane) spectrum (Table 8) of 4 showed a [M+H]⁺ ion at m/z 623,indicating a molecular weight of 622, identical to the molecular weightof 3. The HR FABMS (glycerol) gave a peak for the molecular ion at m/z623.4874 corresponding to the calculated exact mass of 623.4887 for themolecular formula C₃₇ H₆₆ O₇.

As determined with 3, the presence of a terminal α,β-unsaturatedλ-lactone ring, without a 4-OH group (fragment A), and an adjacentbis-THF ring moiety (fragment B) was obvious from the ¹ H- and ¹³ C-NMRspectra of 4 (Table 9) and by NMR spectral comparisons with compound 3.The presence of three OH groups was also recognized by the analysis ofIR, ¹ H-NMR, ¹³ C-NMR, and CIMS spectra of 4 and the CIMS spectrum ofits triacetate derivative (4a). The related stereochemistry in thebis-THF ring moiety was also established as athreo-trans-threo-trans-threo system based on the ¹ H-NMR spectralanalysis of 4a.

The placements of the adjacent bis-THF ring system (fragment B) and thethree hydroxyl groups of 4 along the

                                      TABLE 8                                     __________________________________________________________________________     ##STR11##                                                                    R    M.sup.+ /MH.sup.+                                                                   A    B    C   D   E   F    G    H  I                               __________________________________________________________________________    H    623   295  327* 365*                                                                              257 435*                                             (CIMS)                                                                             605 (a)                                                                             277* (a)                                                                           309 (a)                                                                            347 (a)                                                                           239 (a)                                                   587 (a)                                                                       569 (a)                                                                  TMS  838*  367  471  437     507 331       187                                                                              753                             (EIMS)                                                                             748 (b)                                                                             277* (b)                                                                           381* (b)                                                                           347 (b) 417 (b)                                                                           241 (b)      663 (b)                              658* (b)   291 (b)          151* (b)     573* (b)                             568* (b)                                 483* (b)                        d.sub.9 -TMS                                                                       865*  376  489  446     516 349  669  196                                                                              780*                            (EIMS)                                                                             766 (c)    390* (c)                                                                           347 (c) 417 (c)                                                                           250 (c)                                                                            570 (c)                                      667 (c)    291 (c)          151* (c)                                                                           471* (c)                                     568 (c)                                                                  __________________________________________________________________________     (a): loss of H.sub.2 O (m/z 18);                                              (b): loss of TMSi (m/z 90);                                                   (c): loss of d.sub.9 -TMSi (m/z 99);                                          *weak intensity.                                                              Diagnostic mass fragmentation ions of asiminacin (4) and its triTMSi and      trid.sub.9 -TMSi derivatives.                                            

                                      TABLE 9                                     __________________________________________________________________________    NMR Data for Asiminacin (3) and Its Triacetate derivative (4a)                         .sup.1 H-NMR                                                                  (500 MHz, CDCl.sub.3, δ in ppm,                                                           .sup.13 C-NMR                                               J in Hz)          (125.75 MHZ, CDCl.sub.3,                           H/C No.  4        4a       δ in ppm)                                    __________________________________________________________________________     1       --       --       173.83                                              2       --       --       134.25                                              3       2.26 tt (7.8.1.6)                                                                      2.26 tt (7.8, 1.6)                                                                      31.88-22.67                                       14, 25   1.67 m   1.46-1.64 m                                                                             33.45, 33, 22                                     13, 24   3.40 m   4.85 m    73.89, 74.07                                      16, 23   3.86 m   3.98 m    83.05, 83.19                                      17a, 18a, 21a, 22a                                                                     1.98 m   1.95 m    31.88-22.67                                       17b, 18b, 21b, 22b                                                                     1.67 m   1.79 m    31.88-22.67                                       19, 20   3.96 m   3.90 m    81.78, 81.85                                      27, 29   1.44 m   1.46-1.64 m                                                                             37.31, 37.56                                      28       3.60 m   4.85 m    71.73                                              4-13, 26, 30-33                                                                       1.22-1.72 m                                                                            1.20-1.38 m                                                                             31.88, 29.64, 29.55, 29.41,                                                   29.34, 29.22, 29.04, 29.00,                                                   28.40, 27.42, 25.71, 25.69,                                                   25.21, 21.78, 22.67                               34       0.882 t (7.0)                                                                          0.376 t (7.0)                                                                           14.15                                             35       6.99 q (1.5)                                                                           6.99 q (1.5)                                                                           148.78                                             36       5.00 qq (6.8. 1.7)                                                                     5.00 qq (6.8, 1.7)                                                                      77.40                                             37       1.41 d (6.5)                                                                           1.41 d (7.0)                                                                            19.27                                             13-OAc            2.08 s                                                      24-OAc            2.08 s                                                      28-OAc            2.03 s                                                      __________________________________________________________________________

aliphatic chain were determined based on the fragmentation pattern ofthe triTMSi and tri-d₉ -TMSi derivatives of 4 in their EIMS spectra(Table 8). That the bis-THF ring was located from C-16 to C-23, and thethree hydroxyl groups were situated at the C-15, C-24 and C-28positions. Hence, the only difference between 3 and 4 is that the thirdhydroxyl group is positioned at C-10 in compound 3, while compound 4 hasits third hydroxyl group at C-28. Compound 4 was given the trivial nameasiminacin.

Compound 5 is another waxy compound. A molecular ion peak at m/z 623 inthe CIMS (isobutane) spectrum of 5 (Table 10), indicated a molecularweight of 622. The HR CIMS (isobutane) spectrum showed an exact masspeak at m/z 623.4868 which matched the molecular formula C₃₇ H₆₆ O₇(calculated 623.4887).

The IR, UV, ¹ H-NMR, ¹³ C-NMR spectra of 5 (Table 11) were very similarto those of 3 and 4. Comparisons of these spectra gave the indicationthat 5 could also possess the identical structural skeleton as that ofasimin (3) and asiminacin (4); this included the terminalα,β-unsaturated lactone ring (fragment A), the adjacent bis-THF ringsystem (fragment B), and three hydroxyl groups. The relatedstereochemistry in the bis-THF ring system was defined as having thethreo-trans-threo-trans-threo pattern by the analysis of the usualdiagnostic proton chemical shifts in the ¹ H -NMR spectrum of thetriacetate 5a.

The EIMS diagnostic fragment peaks of the triTMSi and tri-d₉ -TMSiderivatives of 5 (Table 10) determined the location of the adjacentbis-THF ring system at C-16 to C-23 and the two flanking hydroxyl groupsat C-15 and C-24 which were identical to the structures of 3 and 4. Theplacement of the third hydroxyl group at C-29 was suggested by fragmentions at m/z 173 and 767 in the EIMS spectrum of the triTMSi derivativeof 5 and was confirmed by the corresponding diagnostic ions at m/z 182and 794 in the

                                      TABLE 10                                    __________________________________________________________________________     ##STR12##                                                                    R      M.sup.+ /MH.sup.+                                                                   A      B    C     D    E      F   G                              __________________________________________________________________________    H      623   295    365  257*  435*            551                            (CIMS) 605 (a)                                                                             277* (a)                                                                             347 (a)                                                                            239 (a)                                                                             417 (a)                                               587 (a)                                                                       569 (a)                                                                (TMS)  838*  367    437  401*  507  331    173 767                            (EIMS) 748 (b)      347 (b)                                                                            311 (b)                                                                             417 (b)                                                                            241* (b)                                         658* (b)                     151* (b)                                         568* (b)                                                               D.sub.9 -TMS                                                                         865*  376    446  419*  516  349*   182 793                            (EIMS) 766 (c)      347 (c)    417 (c)                                                                            250 (c)    (695)                                 667 (c)                      151 (c)    (596)                                 568 (c)                                 497 (c)                        __________________________________________________________________________     (a): loss of H.sub.2 O (m/z 18);                                              (b): loss of TMSi (m/z 90);                                                   (c): loss of d.sub.9 -TMSi (m/x 99);                                          *weak intensity.                                                              Diagnostic mass fragmentation ions of asiminecin (5) and its triTMSi and      trid.sub.9 -TMSi derivatives.                                            

                                      TABLE 11                                    __________________________________________________________________________    NMR Data for Asiminacin (5) and Its Triacetate derivative (5a)                         .sup.1 H-NMR                                                                  (500 MHz, CDCl.sub.3, δ in ppm,                                                           .sup.13 C-NMR                                               J in Hz)          (125.75 MHZ, CDCl.sub.3,                           H/C No.  4        4a       δ in ppm)                                    __________________________________________________________________________     1       --       --       173.83                                              2       --       --       134.25                                              3       2.26 tt (7.8, 1.6)                                                                     2.26 tt (7.8, 1.6)                                                                      31.94-22.69                                       14, 25   1.68 m   1.46-1.64 m                                                                             33.47, 33.33                                      15, 24   3.40 m   4.85 m    74.07, 73.87                                      16, 23   3.86 m   3.98 m    83.17, 83.05                                      17a, 18a, 21a, 22a                                                                     1.98 m   1.95 m    31.94-22.69                                       17b, 18b, 21b, 22b                                                                     1.68 m   1.78 m    31.94-22.69                                       19, 20   3.86 m   3.90 m    81.82, 81.78                                      28, 30   1.44 m   1.46-1.64 m                                                                             37.52, 37.33                                      29       3.60     4.85      71.81                                              4-13, 26-27,                                                                          1.22-1.72 m                                                                            1.20-1.38 m                                                                             31.94, 29.63, 29.54, 29.34,                       31-33                       29.21, 29.03, 29.02, 28.41.                                                   28.3927.42, 25.70, 25.61,                                                     25.58, 25.38, 22.69                               34       0.891 t (7.0)                                                                          0.880 t (7.0)                                                                           14.11                                             35       6.99 q (1.5)                                                                           6.99 q (1.5)                                                                           148.79                                             36       5.00 qq (6.8, 1.7)                                                                     5.00 qq (6.8. 1.7)                                                                      77.41                                             37       1.41 d (7.0)                                                                           1.41 d (7.0)                                                                            19.26                                             15-OAc            2.08 s                                                      24-OAc            2.08 s                                                      29-OAc            2.04 s                                                      __________________________________________________________________________

EIMS spectrum of the tri-d₉ -TMSi derivative of 5. Isolate 5 was giventhe trivial name asiminecin.

The three novel acetogenins (3-5) are very toxic in the brine shrimptest (BST). This test continues to be a convenient bench-top procedureto discover new cytotoxic compounds. Bioactivity data of compounds 3-5,against three human tumor cell lines, are presented in Table 5. In allcytotoxicity experiments performed human solid tumor cytotoxicity valuesare determined in the same run to eliminate possible variations. Valuesfor adriamycin, as a positive control in the same run, are alsopresented for comparison. In the cytotoxicity experiments conducted byApplicants at the Purdue Cancer Center, 3-5 showed extremely potentcytotoxicities against A-549 (lung cancer), MCF-7 (breast cancer) andH-29 (colon cancer) cell lines. Compounds 3-5 exhibited high selectivityagainst H-29 cancer cells with ED₅₀ values as low as 10⁻¹² μg/ml. Theoverall activities of these three compounds (3-5) are equally or moreactive than the previously reported compounds trilobacin, asimicin andbullatacin and much more active than 4-deoxyasimicin which lacks a thirdhydroxyl. It appears that the substitution of the third hydroxyl groupgreatly enhances the cytotoxic activity. When the third hydroxyl groupis substituted at the C-10, C-28 or C-29 positions, the substitution isequally or more effective in enhancing the cytotoxic activity of thecompound as a substitution at the C-4 position.

Experimental Details for Isolation and Characterization of Compounds 3,4 and 5.

Instrumentation. Mp determinations were made on a Mel-Temp apparatus andare unconnected. Optical rotations were taken on a Perkin Elmer 241polarimeter. IR spectra were obtained on a Perkin-Elmer 1600 FTIRspectrometer. UV spectra were measured on a Beckman DU-7 UVspectrometer. ¹ H and ¹³ C NMR spectra were recorded on a VarianVXR-500S (¹ H at 500 MHZ, ¹³ C at 125.75 MHz) spectrometer in CDCl3 withresonance signals referenced to TMS. Low resolution CIMS, EIMS and FABMS data were collected on a Finnigan 4000 spectrometer. EIMS for TMSiand d₉ -TMSi derivatives and exact mass measurements through peakmatching were performed on a Kratos MS50 mass spectrometer. TLCseparations were made on Si gel 60 F-254 (EM57 17) glass plates (0.25mm) and visualized by spraying with 5% phosphomolybdic acid in EtOH andheating. Chromatotron plates (1 or 2 mm) were prepared with silica gel60 PF 254 containing gypsum and dried at 700 overnight. HPLC was carriedout with a Rainin HPLC instrument using the Dynamax software system anda silica gel column (250×21 mm) equipped with a Rainin UV- 1 detectorset at 220 nm. Chemicals. For preparation of triTMSi and tri-d₉ -TMSiderivatives, N, O-bis-(trimethylsilyl)-acetamide (BSA) and pyridine insilylation grade were purchased from Pierce Chemical Company (USA); d18-bis-(trimethylsilyl)trifluoroacetamide (d18-BSTFA) was from RegisChemical Company (USA) under the brand name Deutero Regis-d 18.

Derivatization. TriTMSi and tri-d₉ -TMSi derivatives were prepared bytreatment of the isolated acetogenins with BSA for the triTMSiderivatives or d₁₈ -BSTFA for the tri-d₉ -TMSi derivatives in thepresence of pyridine. Approximately 10-50 μg of pure compound was placedin a 100 μl conical reaction vial and dried in a vacuum desiccator overP₂ O₅ for 24 hrs. The sample was treated with 2 μl pyridine and 20 μl ofBSA or d¹⁸ -BSTFA and heated at 70° C. for 30 min. The EIMS measurementsof the derivatives were carried out at a resolution of 1500, scanningmass 900-100 at 30 sec./decade.

Bioassays. The brine shrimp (Artemia salina Leach) test (BST) wasperformed as modified. Zhao et al., Phytochemistry, 33, p. 1065 (1993).Seven-day in vitro cytotoxicity tests against human tumor cell lineswere carried out at the Purdue Cancer Center, using standard protocolsfor A-549 (human lung carcinoma), Gu, Z. M.; Fang, X.-P.; Miesbauer, L.R., Smith, D. L.; and McLaughlin, J. L., 30, 31, and32-Hydroxybullatacinones: Bioactive Terminally-Hydroxylated AnnonaceousAcetogenins from Annona bullata., J. Nat. Prod., 1993, 56, 870-876;MCF-7 (human breast carcinoma) McLaughlin, J. L., Chang, C.-J., andSmith, D. L., Simple Bench-Top Bioassays (Brine Shrimp and Potato Discs)for the Discoveries of Plant Antitumor Compounds: A Review of RecentProgress. In Kinghorn, A. D., Balandrin, J. (Eds): "Human MedicinalAgents from Plants", ACS Symposium Series, Washington, D.C., AmericanChemical Society, 1993, pp. 112-137; and HT-29 (human colon carcinoma),Mclaughlin, J.L., Chang, C.-J., Smith, D.L., "'Bench-Top' Bioassays forthe Discovery of Bioactive Natural Products: An Update", in Rahman,Atta-ur- (Ed): "Studies in Natural Product Chemistry", Vol. 9,Amsterdam, Elsevier, 1991, pp. 383-409, with adriamycin as a positivecontrol. The reported values (Table IV) were tabulated from the same runin order to facilitate comparison for the SAR's.

Plant materials for Compounds 3-5. The bark of Asimino triloba (L.)Dunal was collected from stands growing wild at the Purdue HorticulturalResearch Farm. The identification was confirmed by Dr. George R. Parker,Department of Forestry and Natural Resources, Purdue University. Avoucher specimen of the bark is preserved in the pharmacognosyherbarium.

Extraction and purification. The air-dried pulverized stem bark (15 kg)was extracted exhaustively with 95% EtOH and vacuum evaporated to yieldextract FO01 (1645 g) which was partitioned between H₂ O and CH₂ Cl₂(1:1), giving a water soluble fraction (F002, 5 g), CH₂ Cl₂ solublefraction (F003, 1560 g) and an insoluble interface (F004, 80 g). F003was further partitioned between hexane and 90% MeOH aqueous solution andyielded the MeOH fraction (F005, 650 g) and the hexane fraction (F006,905 g).

Directed by the BST bioassay, the most bioactive fraction, F005 (BSTLC₅₀ 7.151×10⁻¹ μg/ml) (200 g) was further fractionated by open columnchromatography on silica gel (8 Kg, 60-200 mesh), eluting withhexane-EtOAc and EtOAc-MeOH gradients; 12 pools were made from thecollected fractions according to their TLC patterns and evaluated by theBST bioassay. The most active pools (P₇ -P₉) were combined (20 g) andsubjected to further repeated separation by silica gel (1000 g, 230-400mesh) column chromatography eluted with MeOH-CHCl₃ gradients. Furtherpurifications of the most bioactive fractions F11-12 (1.2 g, BST LC₅₀3.5×10⁻² μg/ml) were carried out on Chromatotron plates (2 mm thick),eluted with hexane-CH₂ Cl₂ -MeOH (30:20: 0-5). The white wax from thehexane-CH₂ Cl₂ -MeOH fractions was further resolved by HPLC eluted with10% THF in MeOH--hexane gradients to yield the new acetogenins 3-5.

Asimin (3). Colorless wax (12 mg), [α]D+26 (CHCl₃, 1 mg/ml). UV λ_(max)MeOH nm: 215. IR Vλ_(max) film cm⁻¹ : 3437.9 (OH), 2925.1, 2855.3,1751.6 (C═O), 1457.4, 1318.3, 1199.6, 1068.8, 954.0, 870.1. FAB HR-MS(glycerol) m/z: 623.4907 ([M+H]⁺, found), (623.4887, calcd. for C₃₇ H₆₆O₇). CIMS m/z: see Table 7. EIMS (triTMSi derivative) m/z. EIMS (tri-d₉-TMSi derivative) m/z: see Table 7. ¹ H-NMR (500 MHz, CDCl₃): see Table6. ¹³ C-NMR (125.75 MHz, CDCl₃): see Table 6.

Asimin triacetate (3a). Treatment of 3 (4 mg) with AC₂ O -pyridine (atroom temperature, overnight) and subsequent workup gave 3a as a wax. IRV_(max) film cm⁻¹ : 2926, 2855, 1758, 1733, 1459, 1368, 1317, 1241,1068, 1022, 951. CIMS (isobutane) m/z 749 [M+H]+, 689 [MH-CH₃ COOH]+,629 [MH-2CH₃ COOH]+, 569 [MH-3CH₃ COOH]+, 551, 517, 465, 421, 405, 345,327, 219. ¹ H-NMR (500 MHz, CDCl₃): see Table 6.

Asiminacin (4). Colorless wax (12 mg), [α]D+21.1 (CHCl₃, 3.8 mg/ml). UVλ_(max) MeOH nm: 215. IR V_(max) film cm⁻¹ : 3418.1 (OH), 2925.0, 2855.91752.8 (C═O), 1456.7, 1318.6, 1199.5, 1068.9, 953.0, 872.2. FAB HR-MS(glycerol) m/z: 623.4874 ([M+H]+, found), (623.4887, calcd. for C₃₇ H₆₆O₇). CIMS m/z: see Table 8. EIMS (triTMSi derivative) m/z see Table 8.EIMS (tri-d₉ -TMSi derivative) m/z see Table 8. ¹ H-NMR (500 MHz,CDC13): see Table 9. ¹³ C-NMR (125.75 MHz, CDCl₃): see Table 9.

Asiminacin triacetate (4a). Treatment of 4 (4 mg) with AC₂ O -pyridine(at room temperature overnight) and subsequent workup gave 4a as a wax.IR V_(max) film cm⁻¹ : 2929, 2855, 1753, 1732, 1456, 1369, 1317, 1241,1072, 1020, 949. CIMS (isobutane) m/z 749 [M+H]+, 689 [MHCH₃ COOH]+629[MH-2CH₃ COOH]+, 569 [MH-3CH₃ COOH]+, 551, 465, 423, 407, 351, 347, 329,219. ¹ H-NMR (500 MHz, CDCl₃): see Table 9.

Asiminecin (5). Colorless wax (10 mg), [α]D+22 (CHCl₃, 1 mg/ml). UVλ_(max) MeOH nm: 215. IR V_(max) film cm⁻¹ : 3419.4 (OH), 2925.0,2855.9, 1751.3 (C═O), 1455.7, 1317.6, 1198.5, 1068.9, 953.0, 872.2.HR-CIMS (isobutane) m/z: 623.4868 ([M+H]⁺, found), (623.4887, calcd. forC₃₇ H₆₆ O₇). CIMS m/z: see Table 10. EIMS (triTMSi derivative) m/z: seeTable 10. EIMS (tri-d₉ -TMSi derivative) m/z see Table 10. ¹ H-NMR (500MHz, CDCl₃): see Table 11. ¹³ C-NMR (125.75 MHz, CDCl₃): see Table 11.

Asiminecin triacetate (5a). Treatment of 5 (4 mg) with AC₂ O -pyridine(at room temperature, overnight) and subsequent workup gave 5as a wax.IR V_(max) film cm⁻¹ : 2930, 2845, 1754, 1735, 1460, 1370, 1318, 1242,1072, 1024, 953. CIMS (isobutane) m/z 749 [M+H]+, 689 [MHCH₃ COOH]+, 629[MH-2CH₃ COOH]+, 569 [MH-3CH₃ COOH]+, 521, 425, 397, 391, 369, 331, 295,271, 257,217, 201, 169, 101. ¹ H-NMR (500 MHz, CDCl₃): see Table 11.

Goniothalamus giganteus Hook. f. et Thomas (Annonaceae) is a tropicaltree widely distributed in southeast Asia. Extracts of the bark,obtained from Thailand, showed toxicities in the brine shrimp test (BST)and showed murine toxicities in the 3PS (P388) leukemia bioassay. Fromthe ethanol extract of the bark, eleven highly cytotoxic Annonaceousacetogenins have been isolated and, among them, four have a double bondin the aliphatic chain. The presence of a double bond is a relativelyrare feature in the Annonaceous acetogenins. Over 90 acetogenins havebeen described, yet, only one additional acetogenin, bullatenin fromAnnona bullata, has been found having a double bond in the chain. Wehave isolated from the bark of G. giganteus a new mono-THF acetogenin,gonionenin, which also has a double bond in the aliphatic chain. TheC-21/22 double bonds in gonionenin and another mono-THF acetogenin,gigantetronenin (28), previously isolated from this plant, were oxidizedwith m-chloroperbenzoic acid to epoxides which then were cyclized usingperchloric acid with OH groups appropriately located to form another THFring. Gigrantetronenin (28) gave a pair of adjacent bis-THF and a pairof nonadjacent bis-THF (22 and 29) acetogenins. The reactions werefacile and the yields of the products were high. This conversion ofmono-THF acetogenins to adjacent or nonadjacent bis-THF acetogenins notonly conclusively determined the position of the double bonds in themolecules, but also significantly increased the cytotoxic potenciesagainst certain human solid tumor cell lines (Table 12). These reactionsmimic proposed biogenetic pathways leading to the THF rings ofAnnonaceous acetogenins. This is the first reported preparation ofbis-THF Annonaceous acetogenins through epoxidation and cyclization frommono-THF acetogenins possessing a chain double bond.

Gigantetronenin (28) is a major acetogenin isolated from G. giganteusand contains a mono-THF and an isolated chain double bond. Recently, 28was also found in Xylopia ##STR13##

                  TABLE 12                                                        ______________________________________                                        Bioactivities of compounds 28, 29, and 22.                                          BST        A-549     MCF-7    HT-29                                     Comp  LC.sub.50(μg/ml)                                                                      LC.sub.50(μg/ml)                                                                     LC.sub.50(μg/ml)                                                                    LC.sub.50(μg/ml)                       ______________________________________                                        28    10.4       2.22 ×                                                                            1.49 × 10.sup.-8                                                                  .sup. 1.0 × 10.sup.-12                              10.sup.-8                                                    29    3.44 × 10.sup.-2                                                                   <10.sup.-2                                                                               1.0 × 10.sup.-4                                                                 <10.sup.-12                               22    2.49 × 10.sup.-1                                                                   <10.sup.-12                                                                             <10.sup.-12                                                                            <10.sup.-12                               Adria-                                                                                8 × 10.sup.-2                                                                    9.20 ×                                                                            2.31 × 10.sup.-1                                                                 3.92 × 10.sup.-2                    mycin            10.sup.-3                                                    ______________________________________                                         All the cytotoxicities against human tumor cell lines were tested in the      same run; Adriamycin is included as a positive control standard               BST = Brine shrimp lethality test                                             A549 = Human lung carcinoma                                                   MCF7 = Human breast carcinoma                                                 HT29 = Human colon adenocarcinoma                                        

                  TABLE 13                                                        ______________________________________                                        Comp   BST      A-549      MCF-7    HT-29                                     ______________________________________                                        23     57       9.42 × 10.sup.-1                                                                   4.85     1.61 × 10.sup.-2                    Adriamy-                                                                             8 × 10.sup.-2                                                                    8.50 × 10.sup.-3                                                                   5.19 × 10.sup.-1                                                                 3.72 × 10.sup.-2                    cin                                                                           ______________________________________                                         All the cytotoxicities against human tumor cell lines were tested in the      same run; Adriamycin is included as a positive control standard               BST = Brine shrimp lethality test                                             A549 = Human lung carcinoma                                                   MCF7 = Human breast carcinoma                                                 HT29 = Human colon adenocarcinoma    aromatica. In a suggested biogenetic     pathway of the acetogenins from G. giganteus, it has been suggested that     28 was possibly the precursor of gigantecin. To mimic this proposed     biogenesis, 28 was treated with m-CPBA then perchloric acid and produced a     mixture of 29 and 22 (Scheme I) which were separated by HPLC.

The disappearance of double bond NMR signals of 28, the proton signalsat 83.83 (2H, H-18 and 21), 3.47 (H-17), and 3.40 (H-22), and carbonsignals at 6 82.8 (C-21), 82.7 (C-18), 74.3 (C-17 and 22) in 29indicated the formation of a new THF ring, since both the ¹ H and ¹³ CNMR data of the remainder of 29 were almost the same as those of 33. Thecarbon skeleton was confirmed by the EIMS analysis of the TMS derivativeof 29. The proton signals of H-19a and 20a of this new THF were at δ1.98and those of H-19b and 20b were at δ1.66; this suggested a transarrangement for the THF (C- 18/21) and both of the carbon signals of theflanking hydroxyl-bearing carbons were at δ74.3 and indicated the threoconfiguration for both carbon centers C-17/18 and C-21/22. Thus, thestructure of 29 was concluded to be as illustrated. The skeleton andrelative stereochemistries of 29 were exactly the same as those ofgigantecin, and co-TLC of 29 and gigantecin in several different solventsystems always showed them to be inseparable. Gigantecin, with lowcontent, was isolated from G. giganteus previously by our group, was thefirst nonadjacent bis-THF acetogenin to be reported, and showed potentbioactivities in several bioassays. This conversion not only supportsthe hypothesis that gigantetronenin (28) may be the precursor ofgigantecin (29) but also provides a new, more abundant, source of 29.

The carbon skeleton of 22, identical to that of 29, was determined bythe EIMS analysis of the TMS derivative of 22. 22 differs from 29 onlyin the stereochemistry of ##STR14## the newly formed THF ring (C-18/21).The proton signals of the THF methylene protons of 22 at δ1.94 and 1.78(Table 14), instead of at δ1.98 and 1.65 corresponding to the trans THFarrangement, indicated the cis configuration for this THF ring. Thisresult also matched well with the suggested mechanism of thecyclization; since 29 has the trans configuration for this THF, 22should have the cis configuration. The structure of 22 (Scheme I), thus,was determined to be as illustrated; it represents a new type ofnonadjacent bis-THF Annonaceous acetogenin and was named C-18/21-cis-gigantecin.

The bioassay data summarized in Table 12 indicates that the newlyprepared bis-THF compounds are more bioactive than their parentcompounds; the potent bioactivities of 29 and 22 are especiallynoteworthy and approach the levels previously observed with adjacentbis-THF acetogenins such as asimicin, trilobacin, and bullatacin.

Experimental Details for Isolation and Characterization of compounds 22,28 and 29.

Methods. Melting points were determined on a Mettler FP5 hot-stageapparatus and are uncorrected. Optical rotations were determinated on aPerkin Elmer 241 polarimeter. I spectra (film) were measured on aPerkin-Elmer 1600 FTIR spectrometer. UV spectra were taken in MeOH on aBeckman DU-7 UV Spectrometer. ¹ H NMR, ¹ H-¹ H COSY, and ¹³ C NMRspectra were obtained on a Varian VXR-500S spectrometer. Low resolutionMS data were collected on a Finnigan 4000 spectrometer. Low resolutionEIMS for TMS derivatives was performed on a Kratos MS50. HRFABMS weremeasured on the Kratos MS50 spectrometer through peak matching. HPLC wasperformed with a Rainin HPLC using a Dynamax software system and asilica gel column (250×21 mm) equipped with a Rainin UV- 1 detector.Analytical TLC was performed on silica gel plates (0.25 mm) developed

                  TABLE 14                                                        ______________________________________                                        .sup.1 H NMR (500 MHz, CDCl.sub.3) Data.sup.a,b of 29, 22 and 30              [δ ppm (J = Hz)]                                                        Proton     29         22         30                                           ______________________________________                                         3a        2.53 ddd   2.52 ddd   2.56 ddd                                      3b        2.40 ddt   2.40 ddt   2.53 ddt                                      4         3.84 m     3.84 m     5.10 m                                       10         3.88 m     3.88 m     3.85 m                                       11         2.02, 1.52 2.02, 1.50 1.97, 1.44                                   12         1.98, 1.66 1.98, 1.60 1.95, 1.54                                   13         3.80 m     3.80 m     3.95 m                                       14         3.41 m     3.42 m     4.83 m                                       15         1.72, 1.44 1.69, 1.48 1.57 m                                       16         1.72, 1.44 1.72, 1.58 1.57 m                                       17         3.41 m     3.47 m     4.86 m                                       18         3.80 m     3.83 m     3.95 m                                       19         1.98, 1.66 1.94, 1.78 1.90, 1.56                                   20         1.98, 1.66 1.94, 1.78 1.90, 1.56                                   21         3.80 m     3.83 m     3.95 m                                       22         3.41 m     3.40 m     4.86 m                                       23         1.44 m     1.45 m     1.39 m                                       34         0.88 t (7) 0.88 t (7) 0.88 t (7)                                   35         7.19 q (1.5)                                                                             7.19 q (1.5)                                                                             7.08 q (1.5)                                 36         5.06 qq    5.06 qq    5.01 qq                                                 (7, 1.5)   (7, 1.5)   (7, 1.5)                                     37         1.43 d (7) 1.43 d (7) 1.40 d (7)                                   4-OAc      --         --         2.02 s                                       10/14-OAc  --         --         2.07 s                                       13/17-OAc  --         --         2.08 s                                       22-OAc     --         --         2.09 s                                       ______________________________________                                         .sup.a Assignments based on .sup.1 H--.sup.1 H COSY, single relayed COSY,     and double relayed COSY in CDCl.sub.3, TMS as the standard.                   .sup.b Signals of other methylene protons appear between δ1.70 and      1.21 m.                                                                  

                  TABLE 15                                                        ______________________________________                                        .sup.13 CNMR (125 MHz, CDCl.sub.3)Data 29 and 22                              C               29.sup.a                                                                             22.sup.b                                               ______________________________________                                         1              174.5  174.5                                                   2              131.1  131.1                                                   3              33.4   33.4                                                    4              69.9   69.9                                                    5              37.3   37.3                                                    6              25.5   25.5                                                    8              26.2   26.2                                                    9              35.5   35.5                                                   10              79.3   79.2                                                   11              32.4   32.4                                                   12              28.4   28.4                                                   13              82.0   81.9                                                   14              74.3   74.4                                                   17              74.1   74.3                                                   18              82.7   82.7                                                   19              --     --                                                     20              --     --                                                     21              82.7   82.8                                                   22              74.4   74.3                                                   23              33.5   34.2                                                   24              25.6   25.8                                                   32              31.9   31.9                                                   33              22.7   22.7                                                   34              14.2   14.2                                                   35              151.8  151.8                                                  36              78.0   78.0                                                   37              19.2   19.1                                                   ______________________________________                                         .sup.a Signals of other methylene carbons appear approximately at δ     30.0, 29.7, 29.6, 29.4.                                                       .sup.b Signals of other methylene carbons appear approximately at δ     30.6, 29.8, 29.7, 29.6, 29.5, 29.4, 28.4, 28.2, and 28.1                 

using CHC₃ -MeOH (9:1) and hexane-acetone (3:2), respectively, andvisualized with 5% phosphomolybdic acid in EtOH.

Bioassay. The extracts, fractions, and isolates were routinely evaluatedfor lethality to brine shrimp larvae (BST). Cytotoxicities against humansolid tumor cells were measured at the Purdue Cell Culture Laboratory,Purdue Cancer Center, for the A-549 lung carcinoma, MCF-7 breastcarcinoma, and HT-29 colon adenocarcinoma.

Plant Material. The stem bark of G. giganteus (B-826538, PR-50604) wascollected in Thailand in September 1978 under the auspices of Dr. RobertE. Perdue, Medicinal Plant Laboratory, USDA, Beltsville, Md., wherevoucher specimens are maintained.

Extraction and Isolation. The residue of the 95% EtOH crude extract of 4kg of the stem bark was partitioned between H₂ O and CHCl₃ to give an H₂O layer and a CHCl₃ layer. The residue of the CHCl₃ layer waspartitioned between hexane and 10 % H₂ O in MeOH to give a MeOH layer(ca. 100 g of dry residue) and a hexane layer. The MeOH residue, whichrepresented the most active fraction in the BST test (LC₅₀ 15.1 μg/ml),was repeatedly chromatographed over Si gel columns directed by BSTactivity, using gradients of C₆ H₆ /E+OA/MeOH, hexane/EtOAc, and CHCl₃/MeOH and purified by HPLC, using hexane-MeOH-THF (90:9:1), to give awhite wax of gonionenin (31 mg).

Oxidization and Cyclization of Gigantetronenin. Gigantetronenin (28) wasavailable as previously isolated from G. giganteus as described above.To gonionenin (90 mg, in 10 ml of CH₂ Cl₂), was added m-chloroperbenzoicAcid (m-CPBA, 13 mg) and stirred for 1 hour at room temperature. Themixture was washed using 1% NaHCO₃ (5 ml) and H₂ O (2×5 ml), and the CH₂Cl₂ layer was dried in vacuo to give the 21/22-epoxide of 28; to the21/22 epoxide (in 10 ml of CH₂ Cl₂) was added 30% perchloric acid(HClO₄, 5 μl ); the mixture was stirred for another hour at roomtemperature to give a mixture of gigantecin (29) andC-18/21-cis-gigantecin (22). The mixture was washed using 1% NaHCO₃ (5ml) and H₂ O (2×5 ml), and the CH₂ Cl₂ layer was dried in vacuo andresolved by HPLC to give 35 mg of 29 (yield: 38%) and 35 mg of 22(yield: 37%).

Gigantecin (29). White wax, mp 109°-110° C., [α]D+5.3° (c 1.8, MeOH); UVλ_(max) : (MeOH) 212 nm (log E, 3.60), IR V_(max) (film) 3453, 2918,2850, 2360, 2340, 1754, 1728, 1469, 1322, 1059 cm-1, HRFABMS (glycerol)obsd 639.4841 (MH+), calcd for C₃₇ H₆₆ O₈ 708 639.4836. ¹ H and ¹³ CNMR: see Tables 14 and 15, respectively.

C-18/21-cis-gigantecin (22). White wax, mp 89°-90° C., 6.7° (c 1.5,MeOH); UV λ_(max) : (MeOH) 212 nm (log E, 3.74), IR V_(max) : (film)3440, 2918, 2851, 1754, 1726, 1468, 1306 cm⁻¹, HRFABMS (glycerol) obsd639.4854 (MH+), calcd for C₃₇ H₆₇ O₈ 639.4836. ¹ H and ¹³ C NMR: seeTables 14 and 15, respectively.

Acetylations. Compounds 29 and 22 (0.5 mg of each) were mixed withanhydrous pyridine/Ac₂ O at rt overnight and, through the usual workup,gave ca. 0.5 mg of the tetra-acetates 29a and 22a respectively. SeeTable 14.

TMS Derivatizations. Compounds 29 and 22 (ca. 0.3 mg of each) weretreated with N,O-bis-(trimethylsilyl)-acetamide (20 μl ) and pyridine (2μl ) and heated at 70° for 30 min to yield the respective tetra-TMSderivatives 29b and 22b.

An additional acetogenin, Goniocin, was isolated from the ethanolicextracts of the bark of Goniothalamus giganteus after partitionings andrepeated chromatographic separations. Lethality of the fraction to thelarvae of brine shrimp in the brine shrimp lethality test was used toguide the fractionation. The residue of the 95% EtOH crude extract of 4kg of the stem bark was partitioned between H₂ O and CHCl₃. The residueof the CHCl₃ layer was partitioned between hexane and 10% H₂ O in MeOHto give 100 g of dry MeOH residue. The MeOH residue, which representedthe most active fraction in the BST test (LC₅₀ 15.1 μg/ml), wasrepeatedly chromatographed over Si gel columns directed by BST activity,using gradients of C₆ H_(6/) EtOAc/MeOH , hexane/EtOAc, and CHCl₃ MeOHand purified by HPLC over silica gel, using hexane-MeOH-THF (90:9:1), togive a white wax of goniocin.

Goniocin (23) represents a novel heterocyclic ring subclass in theseries of Annonaceous acetogenins. This subclass is the adjacent tri-THFring system. In the case of goniocin (23), the relative stereochemistryof this system is trans, threo, trans, threo, trans, and threo from C-10to C-22. Goniocin (23) is a new prototype for a whole series of possiblevariations, whether they are natural or synthetic; and this new subclassis the tri-THF Annonaceous acetogenins.

On the basis of spectral data (Table 16), Goniocin was suggested to be anew acetogenin possessing a terminal α,β-unsaturated γ-lactone with a4-OH group like gigantecin and gigantetronenin. The molecular formula of23, C₃₇ H₆₄ O₇, having one more index of hydrogen deficiency than thebis-THF acetogenins, indicated that there were three THF rings in 23since no extra double bond proton or carbon signals were observed in theNMR spectra. The adjacent tri-THF moiety in 23 was further identified bysix proton signals at δ3.96-3.78 and six oxygenated carbon signals atδ83.0-79.6 (Table 16). The single carbinol carbon signal at δ74.3(C-22), with its corresponding proton signal at δ3.37 (H-22) which hadcorrelation cross peaks with the proton at δ3.80 (H-21) in the ¹ H--¹ HCOSY spectrum, showed that there was only one OH group in 23 adjacent toa THF ring. The carbon signal for C-10 at δ79.3 demonstrated the absenceof an OH group adjacent to this side of the THF system.

                                      TABLE 16                                    __________________________________________________________________________    .sup.13 C NMR and .sup.1 H NMR Data of 23 and its S- and R-Mosher             Esters.                                                                              .sup.13 C (125 MHz)                                                                   .sup.1 H (500 MHz)      .sup.Δδ H                         23      23 (J in Hz)                                                                            S-MPTA-23                                                                            R-MPTA-23                                                                            δ.sub.S -δ.sub.R           __________________________________________________________________________    37     19.1    1.35 d (6.5)                                                                            1.28   1.31   neg                                    36     78.0    5.06 qq (6.5, 1.5)                                                                      4.86   4.91   neg                                    35     151.9   7.20 q (1)                                                                              6.72   6.97   neg                                     1     174.3   --        --     --     --                                      2     131.1   --        --     --     --                                      3     33.2    2.53 dddd, 2.40 dddd                                                                    2.57   2.64   neg                                     4     69.8    3.85 m    5.31   5.37   R                                       5     37.2    1.47 m    1.65   1.62   pos                                     6-8   29.7-25.4                                                                             1.60-1.20 m                                                                             1.60-1.20                                                                            1.60-1.20                                                                            --                                      9     35.6    1.44 m    1.43   1.42   --                                     10     79.6    3.93 m    3.89   3.87   --                                     11     32.1    2.02m, 1.62 m                                                                           1.96, 1.62                                                                           1.96, 1.63                                                                           --                                     12, 15, 16                                                                           28.5-28.3                                                                             1.96 m, 1.70-1.64 m                                                                     1.88-1.58                                                                            1.92-1.60                                                                            --                                     13, 14, 17, 18                                                                       82.3-81.1                                                                             3.92-3.83 m                                                                             3.92-3.82                                                                            3.98-3.84                                                                            --                                     19     28.5    1.96 m, 1.64 m                                                                          1.88-1.58                                                                            1.92-1.60                                                                            --                                     20     28.8    1.96 m, 1.60                                                                            1.91, 1.49                                                                           2.02, 1.56                                                                           neg                                    21     83.0    3.80 m    4.04   4.05   neg                                    22     74.2    3.37 m    5.05   5.05   R                                      23     33.3    1.38 m    1.62   1.50   pos                                    24     25.6    1.60 -1.20 m                                                                            1.29   1.18   pos                                    25-31  29.7-29.3                                                                             1.60-1.20 m                                                                             1.60-1.20                                                                            1.60-1.20                                     32     31.9    1.60-1.20 m                                                                             1.60-1.20                                                                            1.60-1.20                                                                            --                                     33     22.7    1.28 m    1.28   1.28   --                                     34     14.1    0.879 t (7.0)                                                                           0.880  0.880  --                                     __________________________________________________________________________

The placement of the tri-THF rings at C-10 through C-21 was determinedby the EIMS fragmentation of the bis-TMSi derivative of 23.

The Ha and Hb proton signals of the THF methylenes (C-11, 12, 15, 16,19, and 20) at δ2.02-1.96 and 1.70-1.62, respectively, those of H-13,14, 17, and 18 at δ3.92-3.83, and that of H-22 at δ3.37 suggested thatthe relative stereochemistry around the tri-THF rings wastrans-threo-trans-threo-trans-threo, from C-10 through C-22, bycomparisons with the ¹ H NMR data of model compounds and other knownacetogenins. Both of the absolute configurations of the carbinolstereocenters at C-4 and C-22 were determined to be R by preparation ofMosher ester derivatives (MTPA, Table 16). As the C-4 R and C-36 Srelationship has always been found in the acetogenins, the absolutestereochemistry of 23 was proposed to be 4R, 10S, 13R, 14R, 17R, 17R,18R, 21R, 22R, and 36S, as illustrated.

The level of bioactive potencies (Table 13) of goniocin (23) iscomparable to that of adriamycin but is not at a par with bullatacin,bullatacinone, asimicin and their analogues. Our previous research hasindicated that three hydroxyls (especially with one near the methylterminus of the aliphatic chain) are necessary for enhanced potencywithin the adjacent bis-THF acetogenins subclass. Thus, the introductionof a third hydroxyl into the structure of the tri-THF compounds isanticipated to produce enhanced bioactive potencies. The introduction ofsuch chemical changes to goniocin (23) would be difficult chemically,but the natural compounds very likely exist.

Various cancer cells, infectious organisms, and pests responddifferently to acetogenins that are more or less hydrophobic.Formulations into various deliverable drug and pesticidal productsrequire different levels of hydrophobicity. The increased hydrophobicityof the tri-THF acetogenins (vs. their bis-THF relatives) will enhancetheir applications and formulations in certain instances. Applicationsof these agents include uses as: single primary anticancer,anti-infective, antifungal, and other therapeutic agents, and aspesticidal agents; combination drugs/agents with other acetogeninsand/or other compounds/drugs/agents; and as single or combinationdrug/and/agents against multiple (or single) drug-resistant cancers,other drug resistant diseases, and against resistant pests.

Annonaceous acetogenins are mainly composed of three groups, i.e., theadjacent bistetrahydrofuran (THF), non-adjacent bis-THF, and mono-THFsubclasses. All of the acetogenins in these subclasses have multiplestereogenic centers, and, indeed, some are differentiated from eachother only by their stereochemistries. Consequently, the determinationof the relative and absolute stereochemistries of these stereocentershas become a major concern in the elucidation of the structures of new,as well as previously reported, acetogenin compounds. In addition, thestereochemistries, in many cases, influence the relative potencies andbiological specificities.

Because of their waxy nature, the acetogenins and their derivatives donot readily produce crystals suitable for X-ray crystallographicanalysis. Relative stereochemistries around the THF ring(s) and those ofthe ketolactone moieties have typically been determined by comparisonswith synthetic model compounds of known relative stereochemistry. Theabsolute stereochemistry of none of the Annonaceous acetogenins had beendefined until recently when Mosher ester methodology was applied anddemonstrated to be very helpful. This methodology has been successfullyused to determine the absolute configurations of the carbinol centers ofseveral adjacent bis-THF and mono-THF acetogenins.

The non-adjacent bis-THF acetogenins are the newest subclass of theTHF-bearing Annonaceous acetogenins, and some, e.g., bullatalicin, showpromising in vivo antitumor activities although their potencies are lessthan those of the adjacent bis-THF compounds. Their non-adjacent bis-THFrings have made their structural elucidations and assignments of theirrelative stereochemistries more difficult than the adjacent bis-THF andmono-THF subclasses. Bullatalicin, published in 1989, was the secondmember of this subclass, and its relative stereochemistry and those ofothers in this subclass were clarified by applicants in 1993.

The refined Mosher ester methodology analyzes differences between theproton chemical shifts of S- and R-MTPA esters on both sides of thestereogenic carbinol centers. [Rieser, M. J.; Hui, Y.-H; Rupprecht, J.K.; Kozlowski, J. F.; Wood, K. V.; McLaughlin, J. L.; Hanson, P. R.;Zhuang, A.; Hoye, T. R.; J. Am. Chem. Soc. 1992 , 114, 10203]. However,this procedure cannot be applied to the non-adjacent THF acetogenins,such as bullatanocin (20), bullatalicin (18) and squamostatin A (13)because the hydroxyls between the two THF rings are only two carbonsapart, and the phenyl rings of the Mosher esters interfere with eachother. Additionally, it is not feasible to assign accurately, thecomplicated proton chemical shifts of the per-Mosher esters of thesecompounds. Therefore, until now, the absolute configurations of thenon-adjacent bis-THF acetogenins has not been reported.

Similar problems are also encountered in the other subclasses ofacetogenins that have hydroxyls in close proximity to each other, e.g.,those having a vicinal diol like gigantetrocin A (10) and those having a1,4-diol like goniothalamicin (8). In addition, the absoluteconfigurations of non-THF-flanking carbinol centers, such as the C-28 ofsquamocin (16), have not been solved. ##STR15## Consequently, manyAnnonaceous acetogenins remain undefined stereochemically, and manycontinue to be introduced into the literature without determinations oftheir absolute stereochemistries.

Generally, the bis-THF acetogenins show much higher cytotoxic potenciesthan the mono-THF compounds, and their enhanced potency is also evidentin their inhibition of intact mitochondria. In addition solving theabsolute stereochemistries of the nonadjacent bis-THF acetogenins, wehave also determined if the bioactivities of acetogenins is furtherenhanced as the number of rings is increased, i.e., by conversion of themono-THF acetogenins to bis-ring compounds and the bis-THF compounds totri- or tetra-ring compounds.

Mono-alcohols can be converted into intermolecular formaldehyde acetalsusing chlorotrimethylsilane (Me₃ SiCl ) and dimethyl sulfoxide (Me₂ SO).Bal, B.S. and Pinnick, H. W., Organic Chem. 44, p. 3727 (1979). We havemodified this method and successfully employed it to convert 1,2-, 1,4-and/or 1,5-diols of appropriate acetogenins into cyclic intramolecularformaldehyde acetals (Scheme II). ##STR16## The acetal moiety which isformed connects the diols but does not change the stereochemistries oftheir carbinol centers. Significant differences in the ¹ H NMR spectrabetween the acetal protons in the cis or trans configurations of thecyclic formal derivatives then allows assignment of the relativestereochemistries of the diols in the parent compounds. Furthermore, theerythro configurations in 13, 18 and 26 can be definitely assigned atC-23/24 and not at C-15/16 or C-19/20 after this conversion, since intheir respective acetal derivatives only the THF-flanking hydroxyl atC-24 is left free and the chemical shift of H-24 at δ3.82 clearlyindicates that it is erythro.

Since this method does not affect other isolated hydroxyls, thesegroups, then, are free to be converted into Mosher esters. Consequently,the assignments of the proton chemical shifts affected by these Mosheresters become more feasible, i.e., the formalation decreases the numberof hydroxyls, decreases the number of esters, and, hence, decreases thecomplication of proton signals affected by the Mosher esters in theesterified formal derivatives. With the relative stereochemistriesaround the THF ring(s) already in hand, from comparisons of ¹ H NMRspectra with those of model compounds, the absolute stereochemistries ofall of the stereocenters can then be concluded by analyses of the ¹ HNMR data of S- and R-MTPA esters of the formal derivatives.

We have used the conversion to formaldehyde acetals and the subsequentapplication of the refined Mosher ester methodology, to determine theabsolute stereochemistries of several Annonaceous acetogenins, each ofwhich have 1,2-1,4- and/or 1,5-diol moieties. Thus, for the first time,the absolute configurations of these compounds has been defined for thefollowing compounds: bullatanocin (20), (2,4-cis andtrans)-bullatanocinones (24a and 24b), bullatalicin (18), (2,4-cis andtrans)-bullatalicinones (26a and 26b), squamostatin A (13), squamocin(16), gigantetrocin A (10), and goniothalamicin (8).

Many of the acetal derivatives show enhanced cytotoxicities, as comparedto the parent compound, against human tumor cell lines (see data inTable 17 for comp. 21 vs. 20; 25 vs. 24; 15 vs. 13; and 19 vs. 18).Applicants have also determined that these acetal derivatives showsimilar enhancements in a bioassay that quantifies the inhibition ofoxygen uptake by rat liver mitochondria (see Table 17, RMB data),demonstrating the site of their biological action is identical to thatof their parent acetogenins. Thus, the addition of the acetal rings hasnot created a new, unanticipated mode of cytotoxic action, but ratherenhances the bioactive potencies and selectivities, improvingutilization of many of the natural acetogenins.

Preparation of Cyclic Intramolecular Acetogenins Formaldehyde AcetylDerivatives

To determine the absolute configurations of the Annonaceous acetogeninshaving vicinal 1,2-, 1,4, and/or 1,5-diols, the diols are firstconverted into cyclic formaldehyde acetals.

It has been previously reported (Bal, D.S. and Pinnick, H. W., OrganicChem. 44, p. 3727 (1979)) that monoalcohols can be converted tointermolecular formaldehyde acetals derivatives by mixing equivalentmillimolar concentrations of mono-alcohols, Me₃ SiCl, and Me₂ SO.However, applicants found that adding equivalent millimolarconcentrations of Me₃ SiCl and Me₂ SO to acetogenins having 1,2-1,4-,and/or 1,5-diols, resulted in very low yields of cyclic intramolecularformaldehyde acetal derivatives, and the yields did not increase bylengthening the reaction times. Thin layer chromatography (TLC) showedan abundance of unreacted acetogenins.

By increasing the amount of Me₃ SiCl and Me₂ SO to 2-3-fold foldmillimolar excesses over the starting acetogenins, the yields of theacetal derivatives increased to about 30% after 36-72 hrs reactiontimes. Because the reaction

                                      TABLE 17                                    __________________________________________________________________________    Bioactivities of Compound 8-21, and 24-27.                                                                          RMB                                                                           IC.sub.50                                     BST     A-549   MCF-7   HT-29   (n moles/Lt/mg                          Compound                                                                            LC50 (μg/ml)                                                                       ED50 (μg/ml)                                                                       ED50 (μg/ml)                                                                       ED50 (μg/ml)                                                                       protein)                                __________________________________________________________________________    20    4.30 × 10.sup.-1                                                                5.15 × 10.sup.-10                                                               2.42 × 10.sup.-2                                                                1.66 × 10.sup.-11                                                               50.7                                    21    6.50 × 10.sup.-3                                                                1.43 × 10.sup.-11                                                               2.22 × 10.sup.-1                                                                2.07 × 10.sup.-13                                                               11.3                                    24    2.80 × 10.sup.-1                                                                6.43 × 10.sup.-4                                                                2.93 × 10.sup.-1                                                                2.34 × 10.sup.-8                                                                61.1                                    25    1.50 × 10.sup.-2                                                                6.77 × 10.sup.-5                                                                1.28    7.01 × 10.sup.-13                                                               28.3                                    18    1.20    1.22 × 10.sup.-10                                                               2.62 × 10.sup.-1                                                                6.48 × 10.sup.-8                                                                19.2                                    19    4.90 × 10.sup.-2                                                                1.26 × 10.sup.-13                                                               1.85 × 10.sup.-2                                                                9.10 × 10.sup.-13                                                               29.8                                    26    4.70 × 10.sup.-1                                                                5.62 × 10.sup.-5                                                                1.57 × 10.sup.-3                                                                2.42 × 10.sup.-13                                                               23.0                                    27    3.80 × 10.sup.-2                                                                3.69 × 10.sup.-9                                                                6.39 × 10.sup.-2                                                                2.25 × 10.sup.-13                                                               20.5                                    13    2.80    2.68 × 10.sup.-4                                                                7.20 × 10.sup.-5                                                                2.04 × 10.sup.-11                                                               --                                      14    10.2    1.78    1.00 × 10.sup.-3                                                                2.89 × 10.sup.-7                                                                --                                      15    7.00 × 10.sup.-3                                                                4.87 × 10.sup.-9                                                                1.09    3.03 × 10.sup.-13                                                               --                                      16    2.00 × 10.sup.-2                                                                2.49 × 10.sup.-12                                                               3.03 × 10.sup.-2                                                                1.91 × 10.sup.-13                                                               21.5                                    17    4.40 × 10.sup.-3                                                                2.37 × 10.sup.-10                                                               6.95 × 10.sup.-1                                                                2.89 × 10.sup.-13                                                               421.1                                   10    6.00 × 10.sup.-1                                                                4.52 × 10.sup.-8                                                                3.55 × 10.sup.-4                                                                3.06 × 10.sup.-12                                                               287.1                                   11    --      7.98 × 10.sup.-4                                                                4.61 × 10.sup.-1                                                                7.30 × 10.sup.-13                                                               --                                      12    7.50 × 10.sup.-3                                                                6.95 × 10.sup.-2                                                                2.68 × 10.sup.-1                                                                3.57 × 10.sup.-12                                                               290.3                                    8    37.0    7.11 × 10.sup.-6                                                                5.73 × 10.sup.-6                                                                1.71 × 10.sup.-9                                                                554.8                                    9    5.38 × 10.sup.-2                                                                1.05    4.09 × 10.sup.-4                                                                2.19 × 10.sup.-5                                                                203.1                                   Adriamycin                                                                          8.00 × 10.sup.-2                                                                4.19 × 10.sup.-3                                                                4.01 × 10.sup.-1                                                                3.55 × 10.sup.-2                                                                --                                      Rotenone                                                                            1.23 × 10.sup.-2                                                                --      --      --      34.8                                    __________________________________________________________________________     All of the samples were tested in the same run in each bioassy except in      the MBT, in, which the parent compounds and their respective acetal           derivatives were tested in the same run; Adriamycin and Rotenone are          included as a positive antitumor control standards.                           BST = Brine shrimp lethality test                                             A549 = Human lung carcinoma                                                   MCF7 = Human breast carcinoma                                                 HT29 = Human colon adenocarcinoma                                             RMB = Rat mitochondrial bioassey                                         

produces HC₁, which causes decomposition of the acetogenins, furtherelongation of the reaction time did not increase, but, conversely,decreased the product yields. Some unreacted acetogenins can berecovered when purifying the acetal products.

Formulation of the formaldehyde acetal derivatives.

Bullatanocin (20, Illustration 1), has two nonadjacent THF rings andthree THF flanking hydroxyls; all of the hydroxyls possess the threorelative stereochemical relationships with their respective rings. The ¹H NMR spectra of the S- and R-per-MTPA esters of 20 were too complicatedto permit confident assignments. 20 was treated with excesses of Me₃SiCl and Me₂ SO at room temperature for 48 hrs to give a stable productwhich was identified as the 16,19-formaldehyde acetal derivative (21,yield: 30%). The structure of 21 (Illust. 1) was determined by the ¹ Hand ₁₃ C NMR data and confirmed by the HRFABMS [obsd 651.4828, calcd651.4836, for C₃₈ H₆₇ O₈ (MH+)] and the EIMS fragmentation of itsbis-TMSi derivative. The downfield shifts of two hydroxy methine (H-16and 19) protons from δ3.41 in 20 to δ3.66 and 3.63 in 21 and theappearance of two doublets (J =7.5 Hz) at δ5.26 and 4.63 (the acetalprotons), in the ¹ H NMR spectrum of 21 (Table 18), and a carbon signalat δ95.8 (the acetal carbon), in the ¹³ C NMR spectrum of 21, confirmedthe formation of an acetal ring in 21. These NMR data also indicatedthat the newly formed acetal ring possessed the cis relativeconfiguration, and, thus, revealed either an S/R or an R/S relativeconfiguration between C-16 and C-19. Two hydroxylated methine protons atδ3.84 and 3.39 in the ¹ H NMR spectra of 21 shifted downfield to δ5.31and 5.06 in S-MTPA-21 and to δ5.37 and 5.05 in R-MTPA-21 and furtherdemonstrated that only two hydroxyls, at C-4 and C-24, remained free in21. The assignments of the proton chemical shifts of the Mosher estersof 21 were relatively easy, because the phenyl rings were separated bynineteen carbons, too far apart to interfere with each other. The ¹ HNMR data for the bis-S- and R-MTPA-21 are summarized in Table 18. C-24was assigned to have the R absolute configuration, since the sign ofΔδ_(H) (δ_(S) -δ_(R)) is positive for the chain side, showing relativelymore shielding for this side in R-MTPA-21, and negative for the THF ringside, showing relatively more shielding for this side in S-MTPA-21. Asthe relative stereochemistry from C-12 to C-24 of 20 was already known,the absolute configurations of C-12 (R), C-15 (S), C-16 (S), C-19 (R),C-20 (R), and C-23 (R) were readily concluded. The configuration at C-4was determined to be R, according to the data listed in Table 18, andC-36 was assigned as S, based on the usual ubiquitous 4 R, 36Srelationship. Thus, the absolute configuration of bullatanocin (20) isproposed as illustrated in Illustration 1.

(2,4-Cis and trans)bullatanocinones (24a and 24b, Illustration 1) are apair of ketolactone acetogenins differing from each other only in theconfiguration of the 2,4-bisubstituted-γ-lactone ring. Because of theclose similarity of their structures, such 2,4-cis and trans ketolactoneacetogenins are very difficult to separate, and they are usuallyisolated and reported as a pair of isomers. The structures of 24a and24b are very similar to that of 20 except that they possess theketolactone moiety instead of the α,β-unsaturated γ-lactone ring. Thus,the skeletons and relative stereochemistries around the non-adjacentbis-THF rings of 24a and 24b are the same as those of 20. Compounds 24aand 24b as a mixture, were converted into the formaldehyde acetalderivatives [25a and 25b, yield: 30%, HRFABMS: obsd 651.4836, calcd651.4836, for C₃₇ H₆₇ O₈ (MH+)]. The cis-configuration of the acetalring was indicated by the two doublets (J=7.5 Hz) for the acetal

                                      TABLE 18                                    __________________________________________________________________________    .sup.1 H NMR Data of 21, 19 S- and R-Per-MTPA-21, and S- and                  R-Per-MTPA-19 [δ ppm (∫ = Hz)].                                    Proton                                                                             21    S-21  R-21  Δδ.sub.H (δ.sub.S -δ.sub.R)                           3     19    S-19  R-21  Δδ.sub.H                                                          (δ.sub.S -δ.sub                                                   .R)                            __________________________________________________________________________    37   1.44 d (7.0)                                                                        1.29 d (7.0)                                                                        1.32 d (7.0)                                                                        neg   1.43 d (7.0)                                                                        1.29 d (7.0)                                                                        1.32 d (7.0)                                                                        neg                            36   5.06 qq                                                                             4.36 qq                                                                             4.91 qq                                                                             neg   5.07 qq                                                                             4.36 qq                                                                             4.91 qq                                                                             neg                            35   7.19 q (1.5)                                                                        6.72 q (1.5)                                                                        6.98 q (1.5)                                                                        neg   7.19 q (1.5)                                                                        6.72 q (1.5)                                                                        6.98 q (1.5)                                                                        neg                            3a   2.53 ddtd                                                                           2.60  2.68  neg   2.53 ddtd                                                                           2.60  2.69  neg                            3b   2.40 ddt                                                                            2.39  2.60  neg   2.40 ddt                                                                            2.59  2.60  neg                             4   3.84 m                                                                              5.31 m                                                                              5.37 m                                                                              R*    3.94 m                                                                              5.31 m                                                                              5.37 m                                                                              R*                              5   1.47 m                                                                              1.64 m                                                                              1.62 m                                                                              pos   1.47 m                                                                              1.65 m                                                                              1.62 m                                                                              pos                             6˜10                                                                        1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --    1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --                             11a  1.47 m                                                                              1.47 m                                                                              1.47 m                                                                              --    1.48 m                                                                              1.48 m                                                                              1.48 m                                                                              --                             11b  1.40 m                                                                              1.38 in                                                                             1.38 m                                                                              --    1.42 m                                                                              1.40 m                                                                              1.38 m                                                                              --                             12   3.95 m                                                                              3.94 m                                                                              3.94 m                                                                              --    3.96 m                                                                              3.94 m                                                                              3.94 m                                                                              --                             13a  2.01 m                                                                              2.01 m                                                                              2.01 m                                                                              --    2.00 m                                                                              2.02 m                                                                              2.02 m                                                                              --                             13b  1.64 m                                                                              1.62 m                                                                              1.61 m                                                                              --    1.64 m                                                                              1.64 m                                                                              1.63 m                                                                              --                             14a  1.96 m                                                                              1.93 m                                                                              1.93 m                                                                              --    2.00 m                                                                              1.94 m                                                                              1.94 m                                                                              --                             14b  1.66 m                                                                              1.62 m                                                                              1.61 m                                                                              --    1.63 m                                                                              1.64 m                                                                              1.64 m                                                                              --                             15   3.99 m                                                                              3.98 m                                                                              3.99 m                                                                              --    4.06 m                                                                              4.01 m                                                                              4.00 m                                                                              --                             16   3.66 m                                                                              3.59 m                                                                              3.63 m                                                                              neg   3.62 m                                                                              3.62 m                                                                              3.62 m                                                                              --                             17a, 18a                                                                           1.87 m                                                                              1.82 m                                                                              1.92 m                                                                              neg   1.87 m                                                                              1.89 m                                                                              1.84 m                                                                              pos                            17b, 18b                                                                           1.79 m                                                                              1.70 m                                                                              1.76 m                                                                              neg   1.79 m                                                                              1.76 m                                                                              1.72 m                                                                              pos                            38a  5.26 d (7.5)                                                                        5.21 d (7.5)                                                                        5.22 (7.5)                                                                          neg   5.29 d (7.5)                                                                        5.28 d (7.5)                                                                        5.23 d (7.5)                                                                        pos                            38b  4.63 d (7.5)                                                                        4.56 d (7.5)                                                                        4.56 d (7.5)                                                                        -0    4.64 d (7.5)                                                                        4.62 d (7.5)                                                                        4.57 d (7.5)                                                                        pos                            19   3.63 m                                                                              3.61 m                                                                              3.63 m                                                                              neg   3.62 m                                                                              3.62 m                                                                              3.54 m                                                                              pos                            20   4.01 m                                                                              3.94 m                                                                              4.02 m                                                                              neg   4.00 m                                                                              3.95 m                                                                              3.74 m                                                                              pos                            21a  1.96 m                                                                              1.75 m                                                                              1.93 m                                                                              neg   2.00 m                                                                              1.90 m                                                                              1.92 m                                                                              pos                            21b  1.66 m                                                                              1.63 m                                                                              1.77 m                                                                              neg   1.63 m                                                                              1.67 m                                                                              1.60 m                                                                              pos                            22a  1.97 m                                                                              1.91 m                                                                              2.03 m                                                                              neg   1.90 m                                                                              1.93 m                                                                              1.85 m                                                                              pos                            22b  1.64 m                                                                              1.51 m                                                                              1.59 m                                                                              neg   1.93 m                                                                              1.79 m                                                                              1.70 m                                                                              pos                            23   3.84 m                                                                              4.09 m                                                                              4.08 m                                                                              -0    3.94 m                                                                              4.13 m                                                                              4.05 m                                                                              pos                            24   3.39 m                                                                              5.06 m                                                                              5.05 m                                                                              pos   3.98 m                                                                              5.33 m                                                                              5.29 m                                                                              S*                             25   1.40 m                                                                              1.62 m                                                                              1.47 m                                                                              pos   1.35 m                                                                              1.60 m                                                                              1.62 m                                                                              neg                            26   1.40˜1.20                                                                     1.30 m                                                                              1.14 m                                                                              pos   1.40˜1.20                                                                     1.20 m                                                                              1.30 m                                                                              neg                            27˜33                                                                        1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.15                                                                     --    1.40˜1.20                                                                     1.40˜ 1.20                                                                    1.40˜1.20                                                                     --                             34   0.878 t (7.0)                                                                       0.892 t (7.0)                                                                       0.983 t (7.0)                                                                       --    0.878 t (7.0)                                                                       0.883 t (7.0)                                                                       0.882 t (7.0)                                                                       --                             MeO-1                                                                              --    3.57 s                                                                              3.50 s                                                                              --    --    3.52 s                                                                              3.50 s                                                                              --                             MeO-24                                                                             --    3.55 s                                                                              3.64 s                                                                              --    --    3.54 s                                                                              3.56 s                                                                              --                             Ar--10H                                                                            --    7.63˜7.37                                                                     7.65˜7.36                                                                     --    --    7.63˜7.37                                                                     7.65˜7.36                                                                     --                             __________________________________________________________________________     *Absolute configuration of carbinol center.                              

protons, present at δ5.27 and 4.63, in the ¹ H NMR spectra (Table 19) of25a and 25b. C-24 was determined to have the R configuration fromcomparisons of the ¹ H NMR data of S- and R-MTPA esters of 25a and 25b.These data suggested that 24a and 24b, as expected, have the sameabsolute configurations in the non-adjacent bis-THF moiety as those in20. The (2,4-cis and trans)-ketolactone acetogenins can be chemicallyprepared from the original 4-hydroxylated α,β-unsaturated γ-lactoneacetogenins. In the conversion, the configuration at C-4 will not bechanged. Because the R configuration has been found to be universal inall of the natural 4-hydroxylated acetogenins whose absolutestereochemistries have been revealed, the absolute configurations of C-4in 24a and 24b were, thus, also assigned as 4R. Thus, the absoluteconfiguration of 2,4-cis-bullatanocinone (24a) is proposed to be 2R, 4R,12R, 15S, 16S, 19R, 20R, 23R, and 24R, and that of2,4-trans-bullatanocinone (24b) is proposed to be 2S, 4R, 12R, 15S, 16S,19R, 20R, 23R, and 24R.

Since there was only one free hydroxyl left in 25a and 25b, in the ¹ HNMR spectra of S and R-MTPA esters of 25a and 25b, the shieldingdistance that a single Mosher ester could affect was easily observed.The data listed in Table 19 show that the shielding effect of the phenylring could reach at least eight bonds from the chiral carbon center(C-24). This observation can be very useful in determining the absolutestereochemistries of isolated chain carbinol centers, because, in thosesituations, it is often impossible to differentiate and make chemicalshift assignments of the methylene protons closest to the carbinolcenter. However, the small, but very stable and reproducible, chemicalshift differences of the protons on the oxygenated carbons or theterminal methyl group, located several bonds from the carbinol center,can be

                                      TABLE 19                                    __________________________________________________________________________    .sup.1 H NMR Data of 25a and 25b, 27a and 27b, S- and R-MTPA-25a and          25b,                                                                          and S- and R-MTPA-27a and 27b [δ ppm (J = Hz)]                          Proton                                                                             25a & 25b                                                                           S-25a & 25b                                                                         R-25a & 25b                                                                         Δδ.sub.H (δ.sub.S ˜δ.                           sub.R)                                                                              27a & 27b                                                                           S-27a & 27b                                                                         R-27a & 27b                                                                         Δδ.sub.H                                                          (δ.sub.S ˜.delt                                                   a..sub.R)                      __________________________________________________________________________     2 cis                                                                             3.02  3.02 m                                                                              3.02 m                                                                              --    3.02 m                                                                              3.02 m                                                                              3.02 m                                                                              --                             tran 3.03 m                                                                              3.03 m                                                                              3.03 m                                                                              --    3.03 m                                                                              3.03 m                                                                              3.03 m                                                                              --                              3a cis                                                                            1.48 m                                                                              1.48 m                                                                              1.48 n                                                                              --    1.48 m                                                                              1.48 m                                                                              1.48 m                                                                              --                             trans                                                                              2.23 dddd                                                                           2.23 dddd                                                                           2.23 dddd                                                                           --    2.23 dddd                                                                           2.23 dddd                                                                           2.23 dddd                                                                           --                              3b cis                                                                            2.61 dddd                                                                           2.61 dddd                                                                           2.61 dddd                                                                           --    2.61 dddd                                                                           2.61 dddd                                                                           2.61 dddd                                                                           --                             trans                                                                              1.99 m                                                                              1.99 m                                                                              1.99 m                                                                              --    1.99 m                                                                              1.99 m                                                                              1.99 m                                                                              --                              4 cis                                                                             4.39 dddd                                                                           4.39 dddd                                                                           4.39 dddd                                                                           --    4.39 dddd                                                                           4.39 dddd                                                                           4.39 dddd                                                                           --                             trans                                                                              4.54 dddd                                                                           4.54 dddd                                                                           4.54 dddd                                                                           --    4.54 dddd                                                                           4.54 dddd                                                                           4.54 dddd                                                                           --                              4 cis                                                                             1.76 m                                                                              1.76 m                                                                              1.76 m                                                                              --    1.76 m                                                                              1.76 m                                                                              1.76 m                                                                              --                             trans                                                                              1.71 m                                                                              1.71 m                                                                              1.71 m                                                                              --    1.71 m                                                                              1.71 m                                                                              1.71 m                                                                              --                              5b cis                                                                            1.60 m                                                                              1.60 m                                                                              1.60 m                                                                              --    1.60 m                                                                              1.60 m                                                                              1.60 m                                                                              --                             trans                                                                              1.58 m                                                                              1.58 m                                                                              1.58 m                                                                              --    1.58 m                                                                              1.58 m                                                                              1.59 m                                                                              --                              6˜10                                                                        1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --    1.40˜1.20                                                                     1.40˜1.40                                                                     1.40 ˜1.20                                                                    --                             11a  1.47 m                                                                              1.47 m                                                                              1.47 m                                                                              --    1.48 m                                                                              1.48 m                                                                              1.48 m                                                                              --                             11b  1.40 m                                                                              1.38 m                                                                              1.38 m                                                                              --    1.42 m                                                                              1.40 m                                                                              1.38 m                                                                              --                             12   3.95 m                                                                              3.94 m                                                                              3.94 m                                                                              --    3.94 m                                                                              3.95 m                                                                              3.95 m                                                                              --                             13a  2.01 m                                                                              2.00 m                                                                              2.00 m                                                                              --    2.00 m                                                                              2.02 m                                                                              2.02 m                                                                              --                             13b  1.64 m                                                                              1.62 m                                                                              1.61 m                                                                              --    1.64 m                                                                              1.64 m                                                                              1.63 m                                                                              --                             14a  1.96 m                                                                              1.93 m                                                                              1.93 m                                                                              --    2.00 m                                                                              1.95 m                                                                              1.94 m                                                                              --                             14b  1.66 m                                                                              1.62 m                                                                              1.62 m                                                                              --    1.63 m                                                                              1.65 m                                                                              1.64 m                                                                              --                             15   4.00 m                                                                              3.98 m                                                                              4.01 m                                                                              neg   4.04 m                                                                              4.03 m                                                                              4.00 m                                                                              pos                            16   3.67 m                                                                              3.59 m                                                                              3.63 m                                                                              neg   3.62 m                                                                              3.63 m                                                                              3.61 m                                                                              pos                            17a, 18a                                                                           1.87 m                                                                              1.82 m                                                                              1.92 m                                                                              neg   1.87 m                                                                              1.86 m                                                                              1.33 m                                                                              pos                            17b, 18b                                                                           1.79 m                                                                              1.73 m                                                                              1.78 m                                                                              neg   1.79 m                                                                              1.78 m                                                                              1.71 m                                                                              pos                            38a  5.27 d (7.5)                                                                        5.21 d (7.5)                                                                        5.22 d (7.5)                                                                        neg   5.29 d (7.5)                                                                        5.26 d (7.5)                                                                        5.23 d (7.5)                                                                        pos                            38b  4.63 d (7.5)                                                                        4.56 d (7.5)                                                                        4.56 d (7.5)                                                                        -0    4.64 d (7.5)                                                                        4.59 d (7.5)                                                                        4.57 d (7.5)                                                                        pos                            19   3.43 m                                                                              3.58 m                                                                              3.63 m                                                                              neg   3.62 m                                                                              3.61 m                                                                              3.54 m                                                                              pos                            20   4.01 m                                                                              3.93 m                                                                              4.01 m                                                                              neg   4.01 m                                                                              3.95 m                                                                              3.74 m                                                                              pos                            21a  1.96 m                                                                              1.76 m                                                                              1.93 m                                                                              neg   2.00 m                                                                              1.88 m                                                                              1.80 m                                                                              pos                            21b  1.66 m                                                                              1.62 m                                                                              1.77 m                                                                              neg   1.63 m                                                                              1.66 m                                                                              1.61 m                                                                              pos                            22a  1.97 m                                                                              1.92 m                                                                              2.04 m                                                                              neg   1.90 m                                                                              1.92 m                                                                              1.83 m                                                                              pos                            22b  1.64 m                                                                              1.52 m                                                                              1.57 m                                                                              neg   1.83 m                                                                              1.78 m                                                                              1.70 m                                                                              pos                            23   3.94 m                                                                              4.08 m                                                                              4.08 m                                                                              -0    3.94 m                                                                              4.13 m                                                                              4.05 m                                                                              pos                            24   3.39 m                                                                              5.06 m                                                                              5.05 m                                                                              R*    3.98 m                                                                              5.33 m                                                                              5.28 m                                                                              S*                             25   1.40 m                                                                              1.63 m                                                                              1.47 m                                                                              pos   1.35 m                                                                              1.57 m                                                                              1.62 m                                                                              neg                            26   1.30˜1.20                                                                     1.30 m                                                                              1.14 m                                                                              pos   1.30˜1.20                                                                     1.20 m                                                                              1.30 m                                                                              neg                            27˜33                                                                        1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --    1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --                             34   0.878 t (7.0)                                                                       0.881 t (7.0)                                                                       0.883 t (7.0)                                                                       --    0.878 t (7.0)                                                                       0.883 t (7.0)                                                                       0.881 t (7.0)                                                                       --                             35a cis                                                                            2.61 dd                                                                             2.61 dd                                                                             2.61 dd                                                                             --    2.61 dd                                                                             2.61 dd                                                                             2.61 dd                                                                             --                             trans                                                                              2.67 dd                                                                             2.67 dd                                                                             2.67 dd                                                                             --    2.67 dd                                                                             2.67 dd                                                                             2.67 dd                                                                             --                             35b cis                                                                            3.11 dd                                                                             3.11 dd                                                                             3.11 dd                                                                             --    3.11 dd                                                                             3.11 dd                                                                             3.11 dd                                                                             --                             trans                                                                              3.04 dd                                                                             3.04 dd                                                                             3.04 dd                                                                             --    3.04 dd                                                                             3.04 dd                                                                             3.04 dd                                                                             --                             37   2.20 s                                                                              2.20 s                                                                              2.20 s                                                                              --    2.20 s                                                                              2.20 s                                                                              2.20 s                                                                              --                             MeO-24                                                                             --    3.56 s                                                                              3.64 s                                                                              --    --    3.54 s                                                                              3.56 s                                                                              --                             Ar--5H                                                                             --    7.63˜7.37                                                                     7.65˜7.36                                                                     --    --    7.63˜7.37                                                                     7.65˜7.36                                                                     --                             __________________________________________________________________________     *Absolute configuration of carbinol center.                              

clearly observed in the ¹ H NMR spectra of the respective S- and R-MTPAderivatives.

Bullatalicin (18, Illustration 1) has the same skeleton of contiguousatoms as 20. However, 18 possesses two hydroxyls having threo and onehydroxyl having erythro relative stereochemical relationships with theTHF rings. In 20, all three of the hydroxyls are threo to theirrespective THF rings. When 18 was first published, the erythrorelationship was erroneously assigned at C-15/16. This assignment wassubsequently found to be incorrect by careful study of the COSY spectrumof 18, and the location of the erythro linkage was revised to be atC-23/24 by the use of double relayed COSY spectra and by comparisonswith the structures of other acetogenins from the same source. Since theproton chemical shifts of H-16 and H-19 of 18 were very close to eachother and the off-diagonal crosspeaks between H-16 and H-19 in thedouble relayed COSY spectrum of 18 were weak, we wished to verify this.After 18 was converted into the 16,19-formaldehyde acetal derivative(19, Illustration 1 ), using Me₃ SiCl and Me₂ SO, both proton signals atδ3.41 in 18, which are assigned to the protons on the hydroxylatedcarbons having threo relationships with the THF rings, shifted downfieldto δ3.62 in 19, and the proton signal at δ3.88 in 18, which is assignedto the proton on the hydroxylated carbon having an erythro relationshipwith its THF ring, remained almost unchanged in 19 (Table 18) vs. 18.Thus, the fact that a free hydroxyl having an erythro relationship withits THF ring remained at C-24 in 19, confirmed the placement of theerythro linkage at C-23/24 in 18. Again, the acetal protons in 19,appearing as two doublets at 6 5.29 and 4.64, respectively, suggested anR/S or an S/R relative stereochemical relationship between C-16 andC-19. Bis S- and R-MTPA esters of 19 were then prepared, and their ¹ HNMR data are reported in Table 18. The absolute configurations of C-4and C-24 were determined to be R and S, respectively, by the strikingchange of the signs of the Δδ_(H) (δ_(s) -δ_(R)) values on the two sidesof the chiral centers (Table 18). The data thus demonstrates that 18differs from 20 only in the absolute stereochemistry at C-24 (S in 18and R in 20). The structure of 18, showing the proposed absoluteconfigurations (4R, 12R, 15S, 16S, 19R, 20R, 23R, 24S, 36S), isillustrated in Illustration 1.

(2,4-Cis and trans)-bullatalicinones (26a and 26b, Illustration 1) areanother pair of ketolactone acetogenins, isolated and reported as amixture of the two isomers. As with the relationship betweenbullatanocin (20) and its ketolactones (24a and 24b), 26a and 26bpossess the same skeletons and relative stereochemistries around thenonadjacent bis-THF rings as those of bullatalicin (18). In the ¹ H NMRspectrum of their 16,19-formaldehyde acetal derivatives (27a and 27b,Table 19), the acetal proton signals presented two doublets at δ5.29 and4.64 (J=7.5) and suggested the cis configuration of the newly formedacetal ring. The chemical shift of H-24, the only proton left on anhydroxylated carbon in 27a and 27b, appeared at δ3.88, the same as in19, and this observation placed the erythro linkage at C-23/24 in 26aand 26b. Consequently, the S- and R-MTPA esters of 27a and 27b wereprepared, and the ¹ H NMR and COSY spectra of these esters wererecorded. These data (Table 19) further demonstrated that the influenceof a mono-MTPA, as in the S- and R-MTPA esters of 25a and 25b, couldextend over at least 8 bonds. The Δδ_(H) (δ_(S) -δ_(R)) values (Table19) determined the S absolute configuration at C-24. Thus, asillustrated in Illustration 1, the absolute configuration of2,4-cis-bullatalicinone (26a) was concluded to be 2R, 4R, 12R, 15S, 16S,19R, 20R, 23R, and 24S and that of 2,4-trans-bullatalicinone (26b) wasconcluded to be 2S, 4R, 12R, 15S, 16S, 19R, 20R, 23R, and 24S.

Squamostatin A (13, Illustration 2) was originally published withoutdetermination of any stereochemistry. Its relative stereochemistry wasassigned recently by applicants, but its absolute stereochemistryremained unknown. The C-28 hydroxyl made the direct determination of itsabsolute configuration more complicated because the acetal reaction canconvert both 1,4 and 1,5-diols into acetal rings and complete conversionwould leave no free hydroxyl available in 13 for making Mosher esters todetermine the absolute stereochemistry. However, we predicted that theformation of a seven membered acetal ring from a 1,4-diol should befaster than the formation of an eight membered acetal ring from a1,5-diol. The conversion of 13 to the formaldehyde acetal derivativeswas carefully performed, and the progress was checked by TLC every 4hours. The reaction was terminated after 36 hours when almost all of thestarting material had disappeared and two major products had appeared;the two products were purified by HPLC. The ¹ H NMR data of the twoproducts (Table 20) indicated, just as predicted, that one product wasthe 16,19-mono-formaldehyde acetal derivative (15, Illustration 2) of 13and the other, which was much less polar than 15 on TLC, was the 16,19-and 24,28-bis-formaldehyde acetal derivative (14, Illustration 2) of 13.The two pairs of doublets at δ5.27/4.62 and 5.10/4.56 in the ¹ H NMRspectrum (Table 20) of 14 suggested that both formaldehyde acetal ringspossessed the cis relative configuration, and indicated either an R/R oran S/S relative relationship between C-24 and C-28 and either an R/S oran S/R relative relationship between C-16 and C-19 in 13; the latterindication was confirmed by the ¹ H NMR spectrum of 15, in which theacetal protons also appeared as two doublets at δ5.29 and 4.64 and werealmost the same signals as those of 19. Table 20 lists the ¹ H NMR dataof the S- and R-MTPA esters of 15 and the Δδ_(H) (δ_(S) -δ_(R)) values.

                                      TABLE 20                                    __________________________________________________________________________    .sup.1 H NMR Data of 14 and 15, and S- and R-PER-MTPA-15 [δ ppm (J      = Hz)]                                                                        Proton                                                                              14     15     S-15   R-15   Δδ.sub.N (δ.sub.S                                           .δ.sub.R)                             __________________________________________________________________________    37    1.41 d (7.0)                                                                         1.41 d (7.0)                                                                         1.41 d (7.0)                                                                         1.41 d (7.0)                                                                         --                                          36    5.00 qq                                                                              5.00 qq                                                                              5.00 qq                                                                              5.00 qq                                                                              --                                          35    6.99 q 6.99 q 6.93 q 6.93 q --                                           3    2.26 tt                                                                              2.26 tt                                                                              2.26 tt                                                                              2.26 tt                                                                              --                                           4˜10                                                                         1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      --                                          11    1.50 m 1.50 m 1.50 m 1.50 m --                                          12    3.94 m 3.94 m 3.95 m 3.95 m --                                          13a   2.02 m 2.02 m 2.03 m 2.02 m --                                          13b   1.48 m 1.47 m 1.49 m 1.49 m --                                          14a   1.98 m 2.01 m 1.95 m 1.95 m --                                          14b   1.64 m 1.64 m 1.64 m 1.62 m --                                          15    4.03 m 4.04 m 4.01 m 4.00 m --                                          16    3.62 m 3.62 m 3.63 m 3.60 m pos                                         17a, 18a                                                                            1.82 m 1.82 m 1.86 m 1.92 m pos                                         17b, 18b                                                                            1.68 m 1.76 m 1.77 m 1.72 m pos                                         38a   4.62 d (7.5)                                                                         5.29 d (7.5)                                                                         5.28 d (7.5)                                                                         5.23 d (7.5)                                                                         pos                                         38b   4.62 d C7.5)                                                                         4.64 d (7.5)                                                                         4.61 d (7.5)                                                                         4.56 d (7.5)                                                                         pos                                         19    3.62 m 3.62 m 3.61 m 3.53 m pos                                         20    4.00 m 4.01 m 3.93 m 3.73 m pos                                         21a   1.98 m 2.01 m 1.89 m 1.82 m pos                                         21b   1.64 m 1.65 m 1.66 m 1.60 m pos                                         22a   1.98 m 1.90 m 1.90 m 1.81 m pos                                         22b   1.92 m 1.83 m 1.77 m 1.66 m pos                                         23    3.92 m 3.92 m 4.08 m 3.95 m pos                                         24    3.70 m 3.89 m 5.27 m 5.19 m S*                                          25    1.63, 1.43                                                                           1.48, 1.40                                                                           1.57 m 1.57 m -0                                          26    1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      --                                          27    1.40 m 1.47 m 1.57 m 1.51 m pos                                         28    3.54 m 3.60 m 5.02 m 5.03 m S*                                          29    1.22 m 1.42 m 1.57 m 1.60 m neg                                         30˜33                                                                         1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      --                                          34    0.877 t (7.0)                                                                        0.883 t (7.0)                                                                        0.862 t (7.0)                                                                        0.877 t (7.0)                                                                        neg                                         39a   5.10 d (7.5)                                                                         --     --     --     --                                          39b   4.56 d (7.5)                                                                         --     --     --     --                                          MeO-24                                                                              --     --     3.54 s 3.54 s --                                          MeO-28                                                                              --     --     3.53 s 3.52 s --                                          Ar--10H                                                                             --     --     7.60˜7.37                                                                      7.60˜7.37                                                                      --                                          __________________________________________________________________________     *Absolute configuration of carbinol center.                              

Although the Δδ_(H) (δ_(S) -δ_(R)) of H-25 gave a zero value as it wasadversely affected by the Mosher ester at C-28, the Δδ_(H) (δ_(S)-δ_(R)) values of the protons on the THF ring side of the chiral centerat C-24, as far away as to H-1.6, showed universally positive values.This permitted us to conclude that the absolute configuration at C-24 isS and that at C-28 is also S. As noted above, in conducting the analysesof the ¹ H NMR data of the S- and R-MTPA esters of 25a and 25b and theS- and R-MTPA esters of 27a and 27b, we had observed that the shieldinginfluence of an MTPA moiety could reach at least eight bonds. It was,furthermore, very interesting to find that the Δδ_(H) (δ_(S) -δ_(R))value of the terminal methyls (C-34) of the S- and R-MTPA esters of 15was -7.5 Hz (Table 20) and was affected sufficiently for the applicationof Mosher's methodology; this result similarity suggested an S absoluteconfiguration for C-28.

Squamocin (16, Illustration 2) is an adjacent bis-THF acetogenin whichhas, so far, been reported from four Annonaceous plants. Its relativestereochemistry was determined by an X-ray crystallographic study of aderivative, but its absolute stereochemistry was not solved, since theabsolute configuration at C-28 could not be determined. To solve thisproblem, the hydroxyls at C-24 and C-28 were converted into theformaldehyde acetal (17) whose ¹ H NMR data (Table 21) showed the samerelative stereochemical relationship (R/R or S/S) between C-24 and C-28as that previously revealed by X-ray diffraction. This result alsodemonstrated that the relative stereochemistry of such diols could beaccurately predicted by their acetal derivatives. After the absoluteconfiguration at C-15 was determined to be S by analyses of the ¹ H NMRdata of the S- and R-MTPA esters of 17, these absolute stereochemistriesof the other chiral centers, around the bis-THF rings and at C-28, weredetermined by tracing their relative stereochemistries. Squamocin (16)

                                      TABLE 21                                    __________________________________________________________________________    .sup.1 NMR Data of 17, and S- and R-PER-MTPA-16, and                          S- and R-PER-MTPA-17, [δ ppm (J = Hz)]                                  Proton                                                                             S-16  R-16  Δδ.sub.H (δ.sub.S ˜δ.sub.R)                           17    S-17  R-17  Δδ.sub.H (δ.sub                                             .S ˜δ.sub.R)             __________________________________________________________________________    37   1.41 d (7.0)                                                                        1.41 d (7.0)                                                                        --    1.41 d (7.0)                                                                        1.41 d (7.0)                                                                        1.41 d (7.0)                                                                        --                                   36   5.00 qq                                                                             5.00 qq                                                                             --    5.00 qq                                                                             5.00 qq                                                                             5.00 qq                                                                             --                                   35   6.93 q                                                                              6.93 q                                                                              --    6.93 q                                                                              6.93 q                                                                              6.93 q                                                                              --                                    3   2.26 tt                                                                             2.26 tt                                                                             --    2.26 tt                                                                             2.26 a                                                                              2.26 m                                                                              --                                    4˜12                                                                        1.40˜1.20                                                                     1.4˜1.20                                                                      --    1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --                                   13   1.30 m                                                                              1.13 m                                                                              pos   1.40˜1.20                                                                     1.33 m                                                                              1.14 m                                                                              pos                                  14   1.61 m                                                                              1.46 m                                                                              pos   1.39 m                                                                              1.63 m                                                                              1.50 m                                                                              pos                                  15   5.06 m                                                                              5.03 m                                                                              R*    3.39 m                                                                              5.06 m                                                                              5.05 m                                                                              R*                                   16   4.03 m                                                                              3.99 m                                                                              pos   3.82 m                                                                              4.05 m                                                                              4.045 m                                                                             -0                                   17a  1.92 m                                                                              2.01 m                                                                              neg   1.95 m                                                                              1.89 m                                                                              2.02 m                                                                              neg                                  17b  1.52 m                                                                              1.55 m                                                                              neg   1.62 m                                                                              1.50 m                                                                              1.57 m                                                                              neg                                  18a  1.80 m                                                                              1.97 m                                                                              neg   1.95 m                                                                              1.72 m                                                                              1.92 m                                                                              negs                                 18b  1.65 m                                                                              1.74 m                                                                              neg   1.62 m                                                                              1.61 m                                                                              1.70 m                                                                              neg                                  19   3.79 m                                                                              3.82 m                                                                              neg   3.95 m                                                                              3.79 m                                                                              3.39 m                                                                              neg                                  20   3.79 m                                                                              3.63 m                                                                              pos   3.90 m                                                                              3.84 m                                                                              3.39 m                                                                              neg                                  21a  1.90 m                                                                              1.68 m                                                                              pos   1.95 m                                                                              1.97 m                                                                              1.92 m                                                                              neg                                  21b  1.65 m                                                                              1.56 m                                                                              pos   1.60 m                                                                              1.59 m                                                                              1.70 m                                                                              neg                                  22   1.92 m                                                                              1.75 m                                                                              pos   1.95 m                                                                              1.97 m                                                                              1.91 m                                                                              neg                                  22b  1.69 m                                                                              1.57 m                                                                              pos   1.80 m                                                                              1.71 m                                                                              1.73 m                                                                              neg                                  23   3.96 m                                                                              3.87 m                                                                              pos   3.91 m                                                                              3.90 m                                                                              3.92 m                                                                              neg                                  24   5.21 m                                                                              5.14 m                                                                              S*    3.68 m                                                                              3.61 m                                                                              3.61 m                                                                              --                                   25   1.55 m                                                                              1.55 m                                                                              -0    1.64, 1.44                                                                          1.40 m                                                                              1.42 m                                                                              --                                   26   1.40˜1.20                                                                     1.40˜1.20                                                                     --    1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --                                   27   1.58 m                                                                              1.50 m                                                                              pos   1.62 m                                                                              1.62 m                                                                              1.60 m                                                                              --                                   28   4.99 m                                                                              5.03 m                                                                              S*    3.55 m                                                                              3.54 m                                                                              3.55 m                                                                              --                                   29   1.47 m                                                                              1.53 m                                                                              neg   1.62 m                                                                              1.57 m                                                                              1.57 m                                                                              --                                   30˜33                                                                        1.40˜1.20                                                                     1.40˜1.20                                                                     --    1.40˜1.20                                                                     1.40˜1.20                                                                     1.40˜1.20                                                                     --                                   34   0.860 t (7.0)                                                                       0.875 t (7.0)                                                                       neg   0.878 t (7.0)                                                                       0.877 t (7.0)                                                                       0.878 t (7.0)                                                                       --                                   38a  --    --    --    5.10 d (7.5)                                                                        5.08 d (7.5)                                                                        5.09 d (7.5)                                                                        --                                   38b  --    --    --    4.56 d (7.5)                                                                        5.53 d (7.5)                                                                        5.53 d (7.5)                                                                        --                                   MeO-15                                                                             --    --    --    --    3.56 s                                                                              3.64 s                                                                              --                                   MeO-23                                                                             --    --    --    --    --    --    --                                   Ar--H                                                                              --    --    --    --    7.65˜7.35                                                                     7.65˜7.35                                                                     --                                   __________________________________________________________________________     *Absolute configuration of carbinol center.                              

has the same skeleton and relative configurations around the adjacentbis-THF rings as those of bullatacin and bullatacinone, and the aboveresults showed that 16 also possesses the same absolute configurationwithin this moiety. At C-28, 16 was concluded to be S, and by checkingthe Δδ_(H) (δ_(S) -δ_(R)) value of the terminal methyl (C-34) of the S-and R-per-MTPA esters of 16, a negative value similarly suggested the Sabsolute configuration at C-28. This latter observation, as mentionedabove in the discussion of squamostatin A (13), will be helpful to thestereochemical elucidations of several new acetogenins having a chainhydroxyl close to the terminal methyl (at positions C28 to C-32). Insuch cases, it is not feasible to assign the chemical shifts of themethylene protons that are close to the chain carbinol centers in the ¹H NMR spectra of the Mosher esters of these compounds, but the absoluteconfiguration of such chain carbinol centers can be easily solved bysimply observing the Δδ_(H) (δ_(S) -δ_(R)) values of the terminalmethyls.

Gigantetrocin A (10, Illustration 3) is a mono-THF acetogenin having a1,4,5-triol moiety, and the analyses of the ¹ H NMR spectra of the S-and R-per-MTPA esters of 10 failed to solve the absolute configurationof 10. In addition, the triol group was anticipated to produce a mixtureof formaldehyde acetal derivatives which would complicate thedetermination of the absolute configurations of the carbinol centers.Nevertheless, 10 was treated with excesses of Me₃ SiCl and Me₂ SO for 36hrs. Following the routine procedures, two products were purified byHPLC. Just as predicted, the major product (yield: 35%) was the17,18-formaldehyde acetal derivative (12) of 10, and the minor product(yield: 4%) was the 14,17- formaldehyde acetal derivative (11) of 10,demonstrating that it is much easier to form a five membered acetal ringthan to form a seven membered acetal ring. The increase of molecularweights to 608 in both products (HRFABMS: obsd 609.4730 in 11 and609.4714 in 12 for MH⁺, calcd. 609.4730) and the appearance of theacetal proton signals (at δ5.28 and 4.62 in 11 and at δ4.96 in 12) intheir respective ¹ H NMR spectra confirmed the formation of the acetalmoiety in both 11 and 12. The structures of 11 and 12 were furtherconfirmed by their ¹ H and ¹³ C NMR, COSY, and the EIMS data of theirTMS derivatives. The fragment peak at m/z 299 in the EIMS of 11, inparticular, proved the formation of its acetal at C-14 and C-17, andthat at m/z 309 in the EIMS of 12 placed its acetal at C-17 and C-18.The acetal protons appeared as a doublet at δ5.28 and 4.62 in the ¹ HNMR spectrum of 11 and indicated the cis configuration for the newlyformed acetal ring, and the acetal protons presented a singlet at δ4.96in the ¹ H NMR spectrum of 12 and indicated the trans configuration forthe newly formed, acetal ring. The latter data suggested that thevicinal diol at C-17,18 in 10 possesses a threo configuration. The sameresult was previously obtained by analysis of the ¹ H NMR of theacetonide derivative of 10. The S- and R-MTPA esters of 12 were thenprepared, and C-14 was determined to have the S absolute configuration.Since the relative stereochemical relationship between C-14 and C-17 wasalready revealed to be either S/R or R/S, the absolute configuration atC-17 was concluded to be R. The absolute configuration at C-4 was alsoR, as indicated by the Δδ_(H) (δ_(S) -δ_(R)) values in Table 22. Thestructure of 10, showing the proposed absolute configuration (4R, 10R,13S, 14S, 17R, 18R, and 36S), is illustrated in Illustration 4.

Careful readers may have noticed that the Δδ_(H) (δ_(S) -δ_(R)) value ofH-13 of the S- and R-MTPA esters of 12 (Table 22)

                                      TABLE 22                                    __________________________________________________________________________    .sup.1 H NMR Data of 11 and 12, and S- and R-PER-MTPA-12 [δ ppm (J      = Hz)]                                                                        Proton                                                                              1      12     S-12   R-12   Δδ.sub.N (δ.sub.S                                           .δ.sub.R)                             __________________________________________________________________________    35    1.41 d (7.0)                                                                         1.41 d (7.0)                                                                         1.29 d (7.0)                                                                         1.32 d (7.0)                                                                         neg                                         34    5.06 qq                                                                              5.07 qq                                                                              4.86 qq                                                                              4.91 qq                                                                              neg                                         33    7.19 q 7.19 q 6.72 q 6.98 q neg                                          3a   2.53 ddt                                                                             2.54 ddt                                                                             2.60   2.68   neg                                          3b   2.40 ddt                                                                             2.40 ddt                                                                             2.58   2.60   neg                                          4    3.84 m 3.86 m 5.31 m 5.37 m R*                                           5    1.48 m 1.48 m 1.69 m 1.6S m pos                                          6˜8                                                                          1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      --                                           9    1.53 m 1.53 m 1.47 m 1.43 m pos                                         10    3.96 m 3.88 m 3.88 m 3.78 m pos                                         11a   2.02 m 2.02 m 2.01 m 1.88 m pos                                         11b   1.48 m 1.52 m 1.47 m 1.40 m pos                                         12a   1.96 m 1.98 m 2.02 m 1.96 m pos                                         12b   1.64 m 1.61 m 1.60 m 1.56 m pos                                         13    4.00 q 3.78 q 4.00 q 4.02 q ˜0.02                                 14    3.61 m 3.40 m 5.08 m 5.09 m S*                                          15a   1.89 m 1.86 m 1.79 m 1.90 m neg                                         15b   1.80 m 1.40 m 1.50 m 1.68 m neg                                         16    1.85 m 1.57 m 1.50 m 1.55 m neg                                         17    3.52 m 3.52 m 3.30 m 3.46 m neg                                         18    3.47 m 3.52 m 3.32 m 3.47 m neg                                         36a   5.28 d (7.5)                                                                         4.96 s 4.92 s 4.96 s neg                                         36b   4.62 d (7.5)  4.89 s 4.93 s neg                                         19    1.41 m 1.57 m 1.43 m 1.47 m neg                                         20˜31                                                                         1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      1.40˜1.20                                                                      --                                          32    0.880 t (7.0)                                                                        0.880 t (7.0)                                                                        0.880 t (7.0)                                                                        0.880 t (7.0)                                                                        --                                          MeO-4 --     --     3.51 s 3.50 s --                                          MeO-14                                                                              --     --     3.66 s 3.53 s --                                          Ar--10H                                                                             --     --     7.60˜7.37                                                                      7.60˜7.37                                                                      --                                          __________________________________________________________________________     *Absolute configuration of carbinol center.                              

is -0.02 ppm and not a positive value as it is supposed to be.Nevertheless, the conclusion of the R absolute configuration at C-14seems to be unambiguous since all of the other Δδ_(H) (Δ_(S) -δ_(R))values of the protons on both sides of C-14 consistently showed eitherpositive (on the THF ring side) or negative (on the chain and acetalring side) values. The converse value of H-13 might be caused fromdifferent conformations of the THF and acetal rings in the S- and R-MTPAesters of 12. The Δδ_(H) (δ_(S) -δ_(R)) values of such THF flankingprotons (H-23 in the S- and R-MTPA esters of 21, 25a and 25b, H-16 inthe S- and R-MTPA esters of 17, and H-17 in the S- and R-MTPA esters of9) were found to be about zero if the carbinol centers have a threorelative stereochemical relationship with the THF ring.

The relative stereochemistries of the vicinal diols in Annonaceousacetogenins are usually determined by observing the chemical shifts ofthe acetonyl methyl protons in the ¹ H NMR spectra of their acetonidederivatives, i.e., one singlet at δ1.37 indicates threo, and twoseparate singlets at δ1.43 and 1.33 indicate erythro. The acetonidederivative decomposes very easily, and the decomposition can occurduring the purification procedure or even in the NMR tube in CDCl₃during spectral analyses. In some cases, the proton signals of theacetonyl methyls are not distinct since they are usually overlapped withthe large envelope of methylene proton signals. However, theformaldehyde acetal derivatives are relatively stable, and the acetalproton signals, located downfield from the aliphatic methylene protonsignals, can be easily observed. Thus, formaldehyde acetalderivatization can also be used as a convenient method for determinationof the relative stereochemistry of vicinal diols.

Goniothalamicin (8, Illustraton 3) is a common mono-THF acetogenin,whose absolute stereochemistry has not been previously solved. Aparticular problem in determination of the absolute configuration ofgoniothalamicin is focused on C-10. The formaldehyde acetal derivative(9) was prepared, and the analyses of the Δδ_(H) (δ_(S) -δ_(R)) valuesof the S- and R-MTPA esters of 9 resulted in the determination of theabsolute configuration at C-18 to be 18R; those at C-13, C-14, and C-17were then all solved to be R by tracing their relativestereochemistries. The absolute configuration at C-10 was subsequentlyconcluded to be also 10R, since an R/R or an S/S relative configurationrelationship, between C-10 and C-13, had been revealed by theobservation of the acetal protons which presented as a pair of doubletsat δ5.16 and 4.61 (J =7.5 Hz) in the ¹ H NMR spectrum of 9 (Table 23).The Mosher esters also demonstrated that the absolute configuration atC-4 was R, as usual with all acetogenins that have a 4-OH. The structureof 8, showing the proposed absolute configuration (4R, 10R, 13R, 14R,17R, 18R, and 36S), is illustrated in Illustration 3.

Experimental Detail for Preparation and Characterization of CyclicAcetols.

General Information. Bullatanocin (20), (2,4-cis andtrans)-bullatanocinones (24a and 24b), bullatalicin (18) (2,4-cis andtrans)-bullatalicinones (26a and 26b) squamocin (16), gigantetrocin A(10), and goniothalamicin (8), were available as isolated in ourlaboratory from several plant species in the Annonaceae. Squamostatin A(13) was isolated from Annona squamosa and provided by Bayer AG,Germany.

¹ H NMR, 20 COSY, and ¹³ C NMR spectra, all in CDCl₃, were recorded on aVarian vXR-500S spectrometer. Proton chemical shifts were referenced toTMS (δ0.00) and carbon chemical shifts were referenced to CDCl₃ (δ77.0).All proton chemical shifts >δ2.10 were determined directly

                  TABLE 23                                                        ______________________________________                                        .sup.1 H NMR Data of 9 and S- and R-PER-MPTA-9 [δ ppm (J = Hz]          Proton  9         S-9       R-9     Δδ.sub.H (δ.sub.X                                           ˜δ.sub.R)                     ______________________________________                                        35      1.43 d (7.0)                                                                            1.28 d (7.0)                                                                            1.31 (7.0)                                                                            neg                                       34      5.06 qq   4.86 qq   4.91 qq neg                                       33      7.19 q    6.72 q    6.97 q  neg                                        3a     2.53 ddt  2.60      2.68    neg                                        b      2.40 ddt  2.58      2.60    neg                                        4      3.84 m    5.31 m    5.37 m  R*                                         5      1.47 m    1.65 m    1.61 m  pos                                        6      1.40˜1.20                                                                         1.30 m    1.24 m  pos                                        7˜8                                                                            1.40˜1.20                                                                         1.40˜1.20                                                                         1.40˜1.20                                                                       --                                         9      1.58 m    1.72, 1.55 m                                                                            1.73 m  --                                        10      3.66 m    3.60 m    3.62 m  --                                        11      1.80 m    1.80, 1.66 m                                                                            1.87 m  --                                        12      1.80 m    1.72, 1.54 m                                                                            1.73 m  --                                        13      3.66 m    3.62 m    3.63 m  --                                        14      3.99 q    3.92 q    4.02 q  neg                                       15a     1.98 m    1.75 m    1.92 m  neg                                        b      1.70 m    1.65 m    1.79 m  neg                                       16a     1.99 m    1.91 m    2.03 m  neg                                        b      1.64 m    1.52 m    1.60 m  neg                                       17      3.84 q    4.08 q    4.08 q  ˜0                                  18      3.39 q    5.05 q    5.05 q  R*                                        19      1.40 m    1.60 m    1.48 m  pos                                       20      1.40˜1.20                                                                         1.30 m    1.14 m  pos                                       21˜31                                                                           1.40˜1.20                                                                         1.40˜1.20                                                                         1.40˜1.20                                                                       --                                        32      0.880 t (7.0)                                                                           0.880 t (7.0)                                                                           0.882 t (7.0)                                                                         --                                        36a     5.16 d (7.5)                                                                            5.11 d (7.5)                                                                            5.11 d (7.5)                                                                          --                                         b      4.61 d (7.5)                                                                            4.54 d (7.5)                                                                            4.53 d (7.5)                                                                          --                                        MeO-4   --        3.52 s    3.50 s  --                                        MeO-18  --        3.56 s    3.54 s  --                                        Ar--10H --        7.60˜7.37                                                                         7.60˜7.37                                                                       --                                        ______________________________________                                         *Absolute configuration of carbinol center.                              

from the 1-D ¹ H NMR spectra, whereas, because of the large degree ofoverlap among the resonances, all shifts <δ2.10 were carefully estimatedfrom the centers of the relevant 2D COSY off-diagonal peaks. Massspectral data were obtained on Finnigan 4000 or Kratos MS50spectrometers. All of the reagents are Aldrich products.

Preparation and Purification of Formaldehyde Derivatives.

To Me₃ SiCl (100 mg, in 3 ml of CH₂ Cl₂) was added Me₂ SO (100 mg in 2ml CH₂ Cl₂), and the mixture was allowed to stand at room temperaturefor about one hr until a white precipitate appeared. The CH₂ Cl₂ wasdecanted, and the white precipitate was quickly washed with 1 ml of CH₂Cl₂. To this precipitate, the starting acetogenin (30-60 mg, in 5 ml ofCH₂ Cl₂) was added with stirring at room temperature for 36-72 hrs,until almost all of the starting material had disappeared as determinedby TLC. The mixture was washed using 1% NaHCO₃ (5 ml) and H₂ O (2×5 ml),and the CH₂ Cl₂ layer was dried in vacuo. The products were purified bynormal phase open column chromatography (0.5% MeOH in CHCl₃) or HPLC[5-10% MeOH:THF (9:1) in hexane]. Yields were 25-40% and unreactedstarting materials were often recovered.

Preparation and Purification of Mosher Esters.

To an acetogenin or a formaldehyde derivative of an acetogenin (0.5-1mg, in 0.3 ml of CH₂ Cl₂) were sequentially added pyridine (0.2 ml),4-(dimethylamino)pyridine (0.5 mg), and 25 mg of(R)-(-)-α-methoxy-α-(trifloromethyl)-phenylacetyl chloride. The mixturewas stirred at room temperature for 4 hrs and passed through adisposable pipet (0.6×6 cm) containing silica gel (60-200 mesh) andeluted with 3 ml of CH₂ Cl₂. The CH₂ Cl₂ residue, dried in vacuo, wasredissolved in CH₂ Cl₂ and washed using 1% NaHCO₃ (5 ml) and H₂ O (2×5ml); the CH₂ Cl₂ layer was dried in vacuo to give the S-Mosher esters.Using S-(+)-α-methoxy-α-(trifloromethyl)phenylacetyl chloride gave theR-Mosher esters. Both yields were typically higher than 90%.

TMS Derivatizations.

Compounds (ca. 0.3 mg of each) were treated with N,O-bis(trimethylsilyl)acetamide (20 μl ) and pyridine (2 μl ) and heatedat 70° for 30 minutes to yield the respective tetra-TMS derivatives.

Bioassays.

The brine shrimp lethality test (BST), on newly hatched naupleii, wasperformed in our laboratory as previously described. McLaughlin, Methodsin Plant Biochemistry, Vol. 6, Hostettmann, Academic Press, London, pp.1-33 (1991). Seven day cytotoxicities against human solid tumor cellswere measured at the Cell Culture Laboratory, Purdue Cancer Center, forthe A-549 lung carcinoma, MCF-7 breast carcinoma, and HT-29 colonadenocarcinoma. The respiratory functions of rat liver mitochondria(RMB) were measured polarographically by determination of their rates ofoxygen consumption after the addition of acetogenins or theirderivatives.

Derivatizations of Acetogenins

16,19-Formaldehyde acetal derivative of bullatanocin (21): 60 mg ofbullatanocin (20) was converted into 18 mg of 21 (yield: 30%) as a whitewax; HRFABMS: obsd 651.4828, calcd 651.4836, for C₃₈ H₆₇ O₈ (MH+). ¹ HNMR: see Table 18; ¹³ C NMR δ95.8 (C-38, acetal carbon), 81.0 (C-19),80.3 (C-16).

4,24-TMSi derivative of 21: EIMS m/z (rel. int. %) 624 (12.5), 594(28.6), 563 (11.4), 551 (43.1), 533 (II.I), 521(27.0), 503 (36.3), 485(16.3), 483 (14.8), 473 (18.1), 461 (18.9), 455 (28.8), 420 (22.1), 413(39.0), 407 (22.6), 395(24.6), 391(17.1), 381(53.2), (352(22.9),339(33.1), 335(25.2), 323(33.2) 313(45.7), 309(30.6), 299 (37.3),293(37.9), 291(33.5), 275 (33.8), 273(30.7), 269(44.2), 257(25.8),249(24.1), 239 (29.6), 229 (33.7), 227(35.2), 223(69.4), 199(32.4),191(36.6), 184(69.1), 169(100.0).

4,24-S- and R-MTPA esters of 11: White oil-like; ¹ H NMR: see Table 22.

16,19-Formaldehyde acetal derivatives of (2,4-cis andtrans)-bullatanocinones (25a and 25b): 50 mg of (2,4-cis andtrans)-bullatanocinones (24a and 24b) was converted into 15 mg of 25aand 25b (yield: 30.0%) as a white oil; HPFABMS: obsd 651.4836, calcd651.4836, for C38H6708 (MH+); ¹ H NMR: see Table 22.

24-TMSi derivates of 25a and 25b: EIMS m/z (rel. int. %) 745(100.0,M+Na), 479 (6.6), 449(3.3), 413(6.5), 313(9.8), 309(27.9),291(3.2)243(100-0), 141(27.0).

24-S- and R-MTPA esters of 25a and 25b: White oil-like; ¹ H NMR: seeTable 19.

16,19-Formaldehyde acetal derivative of bullatalicin (19) 80 mg ofbullatalicin (18) was converted into 22 mg of 19 (yield: 27.5%) as awhite wax; HRFABMS: obsd 651.4817, calcd 651.4836, for C₃₈ H₆₇ O₈ (MH⁺);¹ H NMR: see Table 22; ¹³ C NMR δ95.7 (C-38, acetal carbon), 81.7(C-19), 80.3 (C-16).

4,24-TMSi derivative of 19: EIMS m/z (rel. int. %) 624 (26.6), 594(38.3), 563 (13.8), 551(100.0), 533(16.0), 521(35.2), 503(67.2),485(7.0), 455(7.7), 420(8.0), 413 (21.9), 407(9.7), 395(12.0), 391(7.4),381 (δ4.7), 352(11.2), 339(12.6), 313(30.2), 309 (9.5), 293 (12.7),291(15.0), 275 (30.7), 273 (12.0), 269(26.5), 243 (100.0), 229(13.1),227(11.1), 223(16.6), 213(57.2).

4,24-S- and R-MTPA esters of 19: White oil-like; ¹ H NMR: see Table 18.

16,19-Formaldehyde acetal derivatives of (2,4-cis andtrans)-bullatalicinones (27a and 27b): 40 mg of (2,4-cis andtrans)-bullatalicinones (26a and 26b) was converted into 13 mg of 25aand 25b (yield: 32.5%) as a white oil; HRFABMS: obsd 651.4823, calcd651.4836, for C₃₈ H₆₇ O₈ (MH+); ¹ H NMR: see Table 19.

24-TMSi derivatives of 27a and 27b: EIMS m/z (rel. int. %) 722 (16.4%,M+), 479 (4.1), 449(3.6), 413(4.4), 383 (14.0), 367 (33.5), 339 (20.7),313 (13.7), 309 (32.4), 291 (6.4) 269 (16.6), 243 (75.7), 213 (11.7),141 (18.4).

24-S- and R-MTPA esters of 27a and 27b: White oil-like; ¹ H NMR: seeTable 19.

Formaldehyde acetal derivatives of squamostatin A: 9 mg of squamostatinA (13) was converted into 1 mg of 16,19- and 24,28-bis-formaldehydeacetal derivative (14) of 13 [yield: 11.1%, white oil-like, HRFABMS:obsd 663.4852, calcd 663.4836, for C₃₉ H₆₇ O₈ (MH+)] and 2.5 mg of16,19-mono-formaldehyde acetal derivative (15) of 13 [yield: 27.8%,white wax, HRFABMS: obsd 651.4828, calcd 651.4836, for C₃₈ H₆₇ O₈(MH⁺)]; ¹ H NMR: of 14 and 15: see Table 20.

24,28-S- and R-MTPA esters of 15: White oil-like; ¹ H NMR: see Table 20.

24,28-formaldehyde acetal derivative of squamocin (17): 65 mg ofsquamocin (16) was converted into 17 mg of 17 (yield: 28.3%) as a whiteoil; HREABMS: obsd 635.4842, calcd 635.4887, for C₃₈ H₆₇ O₇ (MH⁺); ¹ HNMR: see Table 21.

15-TMSi derivative of 17: EIMS m/z (rel. int. %) 573(5.6), 543(10.1),507(15.2), 455 (79.4), 437 (13.7), 435 (10.9), 367 (100.0), 365 (21.5),361(23.1), 339 (6.5), 309 (13.9), 293(13.2), 269(4.6), 252(19.0),199(7.2), 185(12.4), 169(27.0).

15-S- and R-MTPA esters of 17: White oil-like; ¹ H NMR: see Table 21.

Formaldehyde acetal derivatives of gigantetrocin A: 60 mg ofgigantetrocin A (10) was converted into 2.4 mg of 14,17-formaldehydeacetal derivative (11) of 13 [yield: 4.0 %, white wax, HRFABMS: obsd609.4730, calcd 609.4730, for C₃₆ H₆₅ O₇ (MH+)]and 21 mg of17,18-formaldehyde acetal derivative (12) of 10 [yield: 35.0%, whitewax, HRFABMS: obsd 609.4714, calcd 609.4730, for C₃₆ H₆₅ O₇ (MH+)]; ¹ HNMR: of 11 and 12 see Table 22; ¹³ C NMR of 21693.8 (C-36, acetalcarbon), 81.6 (C-18), 81.5 (C-17).

4,1 8-TMSi derivative of 11: EIMS m/z (rel. int. %) 737(8.8), 707(8.0),δ41(3.8), 617(4.8), 551(3.0), 496(21.2), 461 (22.6), 453 (12.6),426(43.2), 423(7.7), 369(18.0), 363(8.2), 353(100.0), 309(24.6),299(84.2), 263(13.8), 245(19.3), 213(48.2)

4-TMSi derivative of 12 : EIMS m/z (rel. int. %) 753(5.9, MH+),737(6.7), 707 (2.0), 663(5.3), 617(3.2), 426(84.6), 399(7.2), 383(28.7), 369 030), 353(100.0), 336 (10.7), 309(64.5), 295(7.4),281(18.1), 279(18.8), 263(19.7), 245(26.0), 243(18.8), 213 (32.4),184(58.4).

4,14-S- and R-MTPA esters of 12: White oil-like; ¹ H NMR: see Table 22.

10,13-Formaldehyde acetal derivative of goniothalamicin (9): 50 mg ofgoniothalamicin (8) was converted into 16 mg of 9 (yield: 32.0% ) as awhite wax; HRFABMS: obsd 609 .4730, calcd 609.4730, for C₃₆ H₆₅ O₇(MH+); ¹ H NMR: see Table 23, ¹³ C NMR δ95.2 (C-36, acetal carbon), 81.1(C-13), 80.0 (C-10).

4, 18-TMSi derivative of 9: ElMS m/z (rel. int. %) 737(1.2), 707(2.7),641 (5.9), 541(4.2), 524(12.5), 496(8.7), 453 (13.5), 425 (41.0), 423(16.0), 385 (15.4), 383 (8.8), 369(22.8), 353(24.0), 299(87.7),213(33.5).

4,18-S- and R-MTPA esters of 9: White oil-like; ¹ H NMR: see Table 23.

Mitochondrial Inhibition Assay Theory

The oxidative phosphorylation pathway (or electron transport chain) isresponsible for oxidizing NADH to NAD⁺ and producing ATP from ADP andphosphate within the mitochondria. There are five enzyme complexes inthis pathway: NADH-ubiquinone reductase (complex I),succinate-ubiquinone reductase (complex II), ubiquinone-cytochrome creductase (complex III), cytochrome c oxidase (complex IV) and the F₀ F₁-ATPase complex (complex V). The end point of the oxidativephosphorylation pathway is the reduction of oxygen (O₂) to water in themitochondria (thus, it is also called respiration), coupled with thesynthesis of ATP by complex V. State 4 respiration is the oxidation ofsubstrates in the absence of a phosphate acceptor (e.g., ADP). State 3respiration occurs when a phosphate acceptor is present and oxygenutilization increases. The ratio of State 3 respiration to State 4respiration is the respiratory control ratio (RCR).

The process of respiration can be measured by using an oxygen electrodeto detect the amount of oxygen present in the assay cell. If respirationis occurring normally, the concentration of oxygen should slowlydecrease as it is reduced to water. However, if there is some type ofinhibition of oxidative phosphorylation occurring, the oxygen level willno longer decrease at the same rate. This rate of oxygen usage can bevisualized by a chart recorder connected to the oxygen electrode. Atnormal respiration, a decreasing baseline slope will be established.During inhibition, however, the baseline slope should decrease (i.e.,the slope will move closer to zero), since the oxygen present is nolonger being reduced. In other words, the oxygen concentration in theassay cell is no longer decreasing at the same rate as in normalrespiration. Furthermore, inhibition sites at specific complexes can bedetermined by competitive inhibition studies. This is accomplished byaddition of a known inhibitor of, e.g., complex I, followed by the newrespiration-inhibiting compound. If the new compound does not change theeffect of the known inhibitor, it can be assumed that they are acting atthe same site. This can be verified by trying inhibitors at other sitesand by reversing the order, i.e., adding the new compound and then theknown inhibitor.

Materials

Trizma base (Tris[hydroxymethyl]aminomethane), DL-malic acid, L-glutamicacid (monopotassium salt), adenosine 5'-diphosphate (potassium salt;from yeast ATP) and rotenone (6αβ-4'5'-tetrahydro-2,3-dimethoxy-5'β-isopropenyl-furano-(3', 2',8',9)-6H-rotoxen-12-one) were purchased from Sigma Chemical Company. EDTA([ethylenedinitrilo)-tetraacetic acid disodium salt]) was purchased fromFisher Scientific. Sucrose and potassium chloride were purchased fromMallinckrodt, and potassium phosphate (potassium dihydrogen phosphate),monobasic, was purchased from J. T. Baker. The dye and bovine serumalbumin (BSA) for the Bradford protein determination assay werepurchased from Bio-Rad as an assay kit.

Mitochondrial Isolation

The method for isolating rat liver mitochondria essentially followed thepublished methodology. Ahammadsahib et al., Life Sciences, 53, pgs.1113-1120 (1993). The isolation buffer used for the isolation ofmitochondria from rat liver consisted of 4 mM Tris-HC1, 0.5 mM EDTA, and250 mM sucrose at a pH of 7.4. The isolation buffer was kept atrefrigeration temperature at all times by storing on ice during thewhole procedure. The rats were supplied by Sprague-Dawley, were male,and weighed approximately 300-320 grams. They were sacrificed withcarbon dioxide, and the livers were removed and immediately placed inthe isolation buffer on ice. The livers were then rinsed with severalwashings of cold buffer, cut into pieces, and homogenized. Thehomogenates were then centrifuged (Sorvall Superspeed RC2-B, 4° C) for10 minutes at approximately 2000 rpms; the supernatant was nextcentrifuged for 20 minutes at approximately 9000 rpms. The pellet wasreserved and resuspended in 2 ml of assay buffer with a glass pipet. Theassay buffer consisted of 100 mM KC1, 5 mM KH₂ PO₄, 1 mM EDTA, 5 mMglutamate, 1 mM malate, and 20 mM Tris-HCl at a pH of 7.4 and was storedat room temperature.

Protein Determination

The protein concentration of the mitochondrial sample was determinedusing the Bradford Assay. Bovine serum albumin was used as a standard,and three replicates of five different concentrations represented thestandard curve. Three replicates of 1 μl and 2 μl were tested for eachmitochondrial sample, with the result from the 2 μl sample used todetermine the protein content. Absorption was determined at 595 nm usinga Beckman DU-7. Spectrophotometer.

Mitochondrial Inhibition Assay

Respiration was monitored polarographically with an oxygen electrode anda biological oxygen monitor. The mitochondrial assay was initiated byallowing 2.9 ml of assay buffer to equilibrate for approximately 5minutes. The system was kept at a constant temperature of 30° C by awater-bath circulator. The mitochondrial sample (0.1 ml) was added, thesolution was equilibrated for 1 minute, and the electrode was inserted.After the initial State 4 respiration had stabilized (as indicated by aconsistent slope on the chart recorder), 5 μl of a 0.05M ADP solutionwas added, and State 3 and State 4 respirations were allowed tostabilize. Next, 10 μl of the freshly prepared acetogenin solution (in95% ethanol) was injected, and the solution was allowed to equilibratefor 2 minutes. After equilibration, 5 μl of ADP was added, again lettingState 3 and State 4 respirations occur. Once either State 4 hadstabilized or 2 minutes had passed, the recording was discontinued. Twoto three replicates at each concentration of the acetogenin of interestwere conducted, depending on the amount of mitochondrial suspensionavailable. State 3 and State 4 respirations were measured by determiningthe slope of each; if a straight line for State 4 was difficult todetermine, the line was determined as the change of slope just followingState 3 respiration.

We claim:
 1. A substantially pure form of a compound of the formula

    CH.sub.3 (CH.sub.2).sub.3 --R.sub.2 --.sub.5 R.sub.1,

wherein R₁ is a group of the formula ##STR17## and R₂ is a divalentgroup selected from the group consisting of ##STR18## and acetylatedderivatives thereof.
 2. A substantially pure form of a compound of theformula

    CH.sub.3 (CH.sub.2).sub.11 --R.sub.3 --(CH.sub.2).sub.5 R.sub.1 '

wherein R₁ ' is ##STR19## and R₃ is selected from the group consistingof ##STR20## and acetylated derivatives thereof.